K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
S. pyogenes, its virulence, antibiotic, phytochemicalsUniversité Laval
Streptococcus pyogenes (group A β-hemolytic Streptococcus (GABHS)) is the common cause of acute bacterial pharyngitis also known as strep throat or sore throat. Recorded cases of GAS bacterial pharyngitis are 15-36% in children and 5-15% in adults. School aged children in seasons of spring and winter harbor the bacterium up to 20% whereas this rate is much lower in adults. On a global scale, over 616 million new cases of GAS pharyngitis occur every year. Not only is this bacterium responsible for hundred millions of pharyngitis cases but also approximately 10,000 to 15,000 cases of invasive GAS cases are reported annually in the United States, accounting for 10% to 13% mortality rate (http://www.cdc.gov/ncidod/dbmd/abcs/forinvasiveandnoninvasivediseaseincidence). Acute infections can lead to rheumatic fever and post-streptococcal glomerulonephritis (kidney inflammation), which distress children worldwide with disability and death, if antibiotic treatment fails or if the disease is left unattended. Rheumatic fever and rheumatic heart disease are known to be the leading causes of cardiovascular death during the first five decades of life in underdeveloped countries mainly concerning children. Tissues contributing to the GAS nosocomial infections are upper respiratory tract, skin, vaginal and anal area, although latter cases are rare.
GAS has several surface proteins and produces numerous extracellular products that facilitate permeation and successive evasion of the host’s immune system. Streptococcal pharyngitis results from the proliferation of GAS in the pharynx. Virulence associated factors enable S. pyogenes to attach to host tissues, elude the immune response, and spread by penetrating the host tissue layers followed by colonization.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Protein Protein Interactions Of Glycine Oxidase (Thi O)bturne
Project done as a final presentation for Experimental Biochemistry. The project was designed and proposed by me and performed by myself and Lauren Pioppo.
S. pyogenes, its virulence, antibiotic, phytochemicalsUniversité Laval
Streptococcus pyogenes (group A β-hemolytic Streptococcus (GABHS)) is the common cause of acute bacterial pharyngitis also known as strep throat or sore throat. Recorded cases of GAS bacterial pharyngitis are 15-36% in children and 5-15% in adults. School aged children in seasons of spring and winter harbor the bacterium up to 20% whereas this rate is much lower in adults. On a global scale, over 616 million new cases of GAS pharyngitis occur every year. Not only is this bacterium responsible for hundred millions of pharyngitis cases but also approximately 10,000 to 15,000 cases of invasive GAS cases are reported annually in the United States, accounting for 10% to 13% mortality rate (http://www.cdc.gov/ncidod/dbmd/abcs/forinvasiveandnoninvasivediseaseincidence). Acute infections can lead to rheumatic fever and post-streptococcal glomerulonephritis (kidney inflammation), which distress children worldwide with disability and death, if antibiotic treatment fails or if the disease is left unattended. Rheumatic fever and rheumatic heart disease are known to be the leading causes of cardiovascular death during the first five decades of life in underdeveloped countries mainly concerning children. Tissues contributing to the GAS nosocomial infections are upper respiratory tract, skin, vaginal and anal area, although latter cases are rare.
GAS has several surface proteins and produces numerous extracellular products that facilitate permeation and successive evasion of the host’s immune system. Streptococcal pharyngitis results from the proliferation of GAS in the pharynx. Virulence associated factors enable S. pyogenes to attach to host tissues, elude the immune response, and spread by penetrating the host tissue layers followed by colonization.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Protein Protein Interactions Of Glycine Oxidase (Thi O)bturne
Project done as a final presentation for Experimental Biochemistry. The project was designed and proposed by me and performed by myself and Lauren Pioppo.
UV raman evidence of a tyrosine in apo human serum transferrin with a low p k...John Clarkson
J. Clarkson* & D.A. Smith, “UV Raman Evidence of a Tyrosine in Apo-Human Serum Transferrin with a Low pKa that is Elevated upon Binding of Sulfate”, FEBS Letts 503(1), 30-34, 2001.
UV resonance raman study of streptavidin binding of biotin and 2 iminobiotin ...John Clarkson
J. Clarkson*, D.N. Batchelder & D.A. Smith, “UV Resonance Raman Study of Streptavidin Binding of Biotin and 2-iminobiotin: Comparison with Avidin”, Biopolymers (Biospectoscopy), 62, 307-314, 2001.
Volunteers in urban Forestry: Treasure or TroubleLee Mueller
For many cities, volunteers have become an increasingly critical part of urban forestry programs. However, whether you are engaging volunteers as part of your community outreach strategy or to help plant trees, volunteers can require significant amounts of time and resources to manage resources that are not always available. Luckily, if assigned the right projects and provided the right leadership, volunteers can add significant value to your forestry programs. This presentation will provide an overview of the value volunteers can provide, choosing effective volunteer projects, and recruiting volunteers. Specific attention will be given to volunteer training needs, efficient communication, and methods of identifying leaders. Participants can be expected to walk away with an understanding of how to appeal to volunteer leaders, the challenges to volunteer approaches, and
unique ways to keep volunteers engaged and supporting your community's work.
Temperature fields during the development of combustion in a rapid compressio...John Clarkson
J. Clarkson, J.F. Griffiths, J.P. MacNamara, & B.J. Whitaker, “Temperature Fields During the Development of Combustion in a Rapid Compression Machine”, Combustion and Flame, 125, 1162-1175, 2001.
Raman microscopy and x ray diffraction a combined study of fibrillin-rich mic...John Clarkson
J.L. Haston, S.B. Engelsen, M. Roessle, J. Clarkson, E.W. Blanch, C. Baldock, C.M. Kielty & T.J. Wess, “Raman microscopy and X-ray diffraction: A combined study of fibrillin-rich microfibillar elasticity”, J. Biol. Chem., 278(42), 41189-41197, 2003.
Probing the chemistries of flavin ring systems of p hydroxybenzoate hydroxyla...John Clarkson
J. Clarkson, B. Pulfey & P.R. Carey, “Probing the Chemistries of Flavin Ring Systems of p-Hydroxybenzoate Hydroxylase by Raman Difference Spectroscopy”, Biochemistry, 36, 12560-12566, 1997.
Raman study of the polarizing forces promoting catalysis in 4 chlorbenzoyl-co...John Clarkson
J. Clarkson, P.J. Touge, K.L. Taylor, D. Dunaway-Mariano & P.R. Carey, “Raman Study of the Polarizing Forces Promoting Catalysis in 4-Chlorbenzoyl-CoA Dehalogenase”, Biochemistry, 36, 10192-10199, 1997.
The effect of core destabilisation on the mechanical resistance of i27John Clarkson
D.J. Brockwell, G.S. Beddard, J. Clarkson, R.C. Zinober, A.W. Blake, J. Trinick, P.D. Olmsted, D.A. Smith & S.E. Radford, “The Effect of Core Destabilisation on the Mechanical Resistance of I27”, Biophys. J., 83(1), 472-483, 2002.
In-vitro biological activities of the free new H4L ( indole-7-thiocarbohydrazone) ligand and its Ni(II), Pd(II) , Pt(II),
Cu(II), Ag(I), Zn(II) and Cd(II) complexes are screened against two cancerous cell lines, that revealed significant
activity only for [Cu2Cl2(H4L)2(PPh3)2] after 72 h treatment by the highest tested concentrations. The Copper(I)
complex was characterized by X-ray Crystallography and the NMR spectra, whereas it has been confirmed to have
momentous cytotoxicity against ovarian, breast cancerous cell lines (Caov-3, MCF-7). The apoptosis-inducing
properties of the Cu(I) complex have been investigated through fluorescence microscopy visualization, DNA
fragmentation analysis and propidium iodide flow cytometry.
Anticancer Activity of New Di-Nuclear Copper (I) ComplexTaghreed Al-Noor
In-vitro biological activities of the free new H4L ( indole-7-thiocarbohydrazone) ligand and its Ni(II), Pd(II) , Pt(II),
Cu(II), Ag(I), Zn(II) and Cd(II) complexes are screened against two cancerous cell lines, that revealed significant
activity only for [Cu2Cl2(H4L)2(PPh3)2] after 72 h treatment by the highest tested concentrations. The Copper(I)
complex was characterized by X-ray Crystallography and the NMR spectra, whereas it has been confirmed to have
momentous cytotoxicity against ovarian, breast cancerous cell lines (Caov-3, MCF-7). The apoptosis-inducing
properties of the Cu(I) complex have been investigated through fluorescence microscopy visualization, DNA
fragmentation analysis and propidium iodide flow cytometry.
Cloning and Expression of Outer Membrane Protein Omp38 Derived from Aeromonas hydrophila in Escherichia coli
http://dx.doi.org/10.21276/SSR-IIJLS.2019.5.3.8
Ultraviolet resonance raman study of the avidin biotin complexJohn Clarkson
J. Clarkson*, C. Sudworth, S.I. Masca, D.N. Batchelder & D.A. Smith, “Ultraviolet resonance Raman study of the avidin biotin complex”, J. Raman Spectrosc., 31, 373-375, 2000.
Opportunities for probing the structure and mechanism of porphobilinogen synt...John Clarkson
J. Clarkson, E.K. Jaffe, R.M. Petrovich, J. Dong & P.R. Carey, “Opportunities for Probing the Structure and Mechanism of Porphobilinogen Synthase by Raman Spectroscopy.” J. Am. Chem. Soc., 119, 11556-11557, 1997.
Morphological study of aluminum tris(8 hydroxyquinoline) thin films using inf...John Clarkson
R.J. Curry, W.P. Gillin, J. Clarkson & D.N. Batchelder, “Morphological study of aluminum tris(8-hydroxyquinoline) thin films using infrared and Raman spectroscopy”, J. Appl. Phys., 92 (4), 1902-1905, 2002.
Evidence for electrophilic catalysis in the 4 chlorobenzoyl-co a dehalogenase...John Clarkson
K.L. Taylor, R-Q. Liu, P-H. Liang, J. Price, D. Dunaway-Mariano, P.J. Tonge, J. Clarkson & P.R. Carey, “Evidence for Electrophilic Catalysis in the 4-Chlorobenzoyl-CoA Dehalogenase Reaction: UV, Raman, and 13C-NMR Spectral Studies of Dehalogenase Complexes of Benzoyl-CoA Adducts”, Biochemistry, 34, 13881-13888, 1995.
Comparison of the structures and vibrational modes of carboxybiotin and n car...John Clarkson
J. Clarkson & P.R. Carey, "Comparison of the Structures and Vibrational Modes of Carboxybiotin and N-Carboxy-2-imidazolidone" J. Phys. Chem. A, 103, 2851-2856, 1999.
Characterization of the surface of a citrate reduced colloid optimized for us...John Clarkson
C.H. Munro, W.E. Smith, M. Garner, J. Clarkson & P.C. White, “Characterization of the Surface of a Citrate-Reduced Colloid Optimized for Use as a Substrate for Surface-Enhanced Resonance Raman Scattering” Langmuir, 11, 3712-3720, 1995.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
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Expression, purification and spectroscopic characterization of the cytochrome p450 cyp121 from mycobacterium tuberculosis
1. Journal of Inorganic Biochemistry 91 (2002) 527–541
www.elsevier.com/locate/jinorgbio
Expression, purification and spectroscopic characterization of the
cytochrome P450 CYP121 from Mycobacterium tuberculosis
a b c d a
Kirsty J. McLean , Myles R. Cheesman , Stuart L. Rivers , Alison Richmond , David Leys ,
d c e f e
Stephen K. Chapman , Graeme A. Reid , Nicholas C. Price , Sharon M. Kelly , John Clarkson ,
e a ,
*W. Ewen Smith , Andrew W. Munro
a
Department of Biochemistry, The Adrian Building, University of Leicester, University Road, Leicester LE1 7RH, UK
b
Centre for Metalloprotein Spectroscopy and Biology, University of East Anglia, Norwich, UK
c
Institute of Cell and Molecular Biology, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
d
Department of Chemistry, University of Edinburgh, The King’s Buildings, West Mains Road, Edinburgh EH9 3JJ, UK
e
Department of Pure and Applied Chemistry, University of Strathclyde, The Thomas Graham Building, Cathedral Street, Glasgow G1 1XL, UK
f
Division of Biochemistry and Molecular Biology, Faculty of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow,
Glasgow G12 8QQ, UK
Received 12 March 2002; received in revised form 23 April 2002; accepted 8 May 2002
Abstract
The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and
the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a
cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins
(major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which
binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein
to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region
describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from
spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole,
miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.
2002 Elsevier Science Inc. All rights reserved.
Keywords: CYP121; P450; Mycobacterium tuberculosis; Azole inhibitors; Drug targets
1. Introduction known to contain a complex lipid-rich outer envelope, a
large proportion of the genome encodes enzymes involved
The pathogenic bacterium Mycobacterium tuberculosis in lipid metabolism. One of the most remarkable findings
represents an enormous threat to human health. It is was the high number of cytochrome P450-encoding (CYP)
responsible for more deaths world-wide than any other genes, of which there are 20. In all bacterial genomes
infectious agent, and is the major cause of death for sequenced before that of Mtb, there are far fewer CYP
HIV-infected individuals in Africa and Asia [1]. A disturb- genes. For instance, neither Escherichia coli or Salmonella
ing factor associated with the global resurgence of tuber- typhi contain any P450s [3,4]. Until the Mtb genome
culosis (TB) is the proliferation of drug- and multi drug- sequence was reported, Bacillus subtilis (with seven) had
resistant strains. Thus, resistance to the traditional drugs the largest number of P450 enzymes in a prokaryote [5].
used to treat TB causes a major problem. Subsequent to the Mtb genome sequence, only other
The genome sequence of M. tuberculosis (Mtb) was actinomycetes (e.g. Streptomyces coelicolor and other
determined in 1998 [2]. As was suspected for a bacterium mycobacteria) have been revealed to encode similarly high
numbers of CYP genes [6,7]. However, the genome of the
leprosy pathogen M. leprae retains only one functional*Corresponding author. Tel.: 144-116-252-3464; fax: 144-116-252-
cytochrome P450 (although there are several pseudogenes)3630.
E-mail address: awm9@leicester.ac.uk (A.W. Munro). [8]. The requirement for the large number of P450s in Mtb
0162-0134/02/$ – see front matter 2002 Elsevier Science Inc. All rights reserved.
PII: S0162-0134(02)00479-8
2. 528 K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541
is unclear, although it seems likely that many are involved encodes a protein with 25% or more identity to several
in pathways that lead to the synthesis of the complex fatty acid hydroxylase P450s from the CYP4A family [16].
mycolipids that provide a robust barrier around the cell The product of Mtb gene Rv2276 (CYP121) shows
[9,10]. Mtb may also contain a sterol biosynthetic path- approx. 28% amino acid sequence identity with Strep-
way, and previous studies have reported the expression and tomyces griseolus CYP105A1 (involved in detoxification
characterization of a P450 with sterol demethylase activity of sulfonylurea herbicides [17]. However, it also has a
[11–13]. This P450 (the product of the Rv0764c gene) is a similar level of identity (29%) with the structurally
member of the CYP51 family of P450s [14]. It is a characterized P450 eryF (CYP107A1) from Sac-
homologue of lanosterol demethylase P450s found in charopolyspora erythraea (Fig. 1) which catalyses the
various yeasts and fungi, which generate the essential hydroxylation of the polyketide 6-deoxyerythronolide B, a
membrane sterol ergosterol, and which is the target for the late step in the synthesis of the antibiotic erythromycin
azole class of antifungal drugs [15]. [18]. It also shows similarity to a variety of other bacterial
Of the 19 other P450s encoded in the Mtb genome, few P450s, including P450 BioI (involved in biotin synthesis in
are of obvious substrate selectivity based on sequence B. subtilis) [19]. Thus CYP121 may play a role in lipid or
comparisons. The majority are most strongly related to polyketide metabolism.
other P450s in the pathogen, perhaps indicating that they In this paper, we report the cloning and expression of
are involved in metabolism of substrates peculiar to the the CYP121 gene from Mtb, leading to the purification and
genus, and that they have arisen by gene duplication and biophysical characterization of the CYP121 P450 enzyme.
diversification of function. Of the other P450s, only the The P450 exhibits several interesting features, including
product of the Rv1394c (CYP132) gene shows sufficient unique spectroscopic properties and strong stabilization of
similarity to other P450s of established function to permit the low-spin form. CYP121 also displays high affinity for
an accurate guess as to its substrate selectivity. CYP132 azole antifungal drugs, which bind to the CYP121 heme
Fig. 1. Amino acid sequence alignment for M. tuberculosis CYP121 (CYP121) and Saccharopolyspora erythraea CYP107A1 (ERYF) involved in
synthesis of the polyketide antibiotic erythromycin. P450 eryF has 28.7% amino acid sequence identity with CYP121, and is the most closely related P450
for which there is atomic structural data. Pairwise alignment was carried out using the ClustalW program via the European Bioinformatics Institute web site
(http://www2.ebi.ac.uk/clustalw/). Highlighted residues in black are conserved cysteine and phenylalanine amino acids essential for heme ligation and
control of redox properties, respectively [29,49]. Highlighted residues in grey are key amino acids for the binding of ketoconazole in P450 eryF, and their
corresponding residues in CYP121 (see Discussion section). Amino acid residues indicated by ‘*’ are identical in both sequences, whereas those with ‘:’
and ‘.’ are ‘highly similar’ and ‘similar’, respectively.
3. K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541 529
iron. This suggests that the enzyme may be a viable drug and re-centrifuged. The final cell pellet was frozen at
target in the battle against multi drug-resistant M. tuber- 220 8C until use.
culosis. The cells were thawed and resuspended in a small
volume of buffer A containing PMSF and benzamidine
hydrochloride (both 1 mM) to minimise proteolysis. Cells
2. Experimental were broken by sonication using a Bandelin Sonoplus
GM2600 sonicator (10320-s bursts at full power on ice,
2.1. Molecular biology with appropriate cooling time between bursts), and the
extract was then passed through a French press (950 p.s.i.,
The Rv2276 gene encoding the putative cytochrome three passes) to complete the cell breakage process. The
P450 from M. tuberculosis (CYP121) was amplified from lysate was centrifuged at 18 0003g for 30 min (at 4 8C)
the cosmid DNA (MTCY339) obtained from Professor and the soluble extract was decanted. Protein was fraction-
Stewart Cole at the Pasteur Institute, Paris. Primers used in ated by ammonium sulfate precipitation (two steps: 0–30
the reaction were designed from the genomic sequence [2], and 30–70%), and the 30–70% pellet (containing
and were as follows: Forward: 59-TATGAC- CYP121) was retained. The pellet was resuspended in a
TCATATGACCGCGACCGTTCTGCTCG-39. The letters minimal volume of buffer A containing 0.5 M ammonium
]]]
underlined indicate an engineered restriction site for NdeI, sulfate. The solution was then loaded onto a Phenyl-
including the initiation codon ATG. Reverse: 59-AAGAC- Sepharose column (5330 cm), pre-equilibrated in buffer A
GGATCCTACCAGAGCACCGGAAGG-39. Underlined containing 1.5 M ammonium sulfate (buffer B). The
]]]
letters show a site for BamHI and the stop codon TAG is protein was eluted in a linear gradient (500 ml) of buffer B
also incorporated. to buffer A. CYP121-containing fractions were dialysed
The PCR fragment of Rv2276 was cloned directly into against 5 l of buffer A, and loaded onto a Q-Sepharose
the pGEM-T vector (Promega, Southampton, UK), creat- column (5325 cm) pre-equilibrated in buffer A. Protein
ing plasmid clone pKM2a. The DNA sequence of Rv2276 was eluted in a linear gradient (500 ml) of buffer A to
was verified at this stage by automated dideoxy DNA buffer A plus 500 mM potassium chloride. CYP121-con-
sequencing (Applied Biosystems DNA sequencer). After taining fractions of highest purity were pooled and
digestion with NdeI and BamHI, the Rv2276 fragment was dialysed against 5 l of 25 mM potassium phosphate (pH
ligated into the T7lac promoter vector pET11a (Novagen, 6.5, buffer C) before being loaded onto a hydroxyapatite
Nottingham, UK) pre-digested with the same restriction column (5325 cm) pre-equilibrated in the same buffer.
endonucleases. The ligation mix was transformed into E. Protein was eluted in a linear gradient (500 ml) of buffer C
coli strain TG1 and plasmid preparations made from to 500 mM potassium phosphate (pH 6.5). Fractions of
transformant colonies, leading to identification of Rv2276 highest purity (A /A .1.5) were retained, pooled,416 280
clones by restriction digests. The final expression plasmid concentrated by ultrafiltration (Centriprep 30, Millipore) to
clone was named pKM2b. All molecular biology was ,2 ml and exchanged into buffer A plus glycerol (50%
performed using standard protocols [20]. v/v) by dialysis, prior to storage of the pure enzyme at
280 8C.
2.2. Expression and purification of CYP121
2.3. Assessment of protein purity and concentrationThe expression plasmid pKM2b was transformed into a
variety of T7 RNA polymerase strains for investigation
Purity of the CYP121 protein was assessed by spectraland optimisation of protein expression. Various growth
properties (ratio of P450-specific absorption at 416.5 nmtemperature and induction conditions were investigated in
compared with protein-specific absorption at 280 nm), andeach case to optimise protein yield and enzyme solubility.
by SDS–PAGE gel electrophoresis of protein samples (onHMS174 (DE3) (Novagen) was ultimately selected for
10% polyacrylamide denaturing gels) at different stages inbest protein expression. Conditions used for CYP121 (the
the purification process. Determination of cytochromeprotein product of gene Rv2276) expression from
P450 concentration was by the method of Omura and SatoHMS174(DE3)/pKM2b transformants were growth at
21
[21], using an extinction coefficient of ´ 591 mM30 8C with vigorous agitation (250 rpm) until the OD of 450-490600
21
the culture (usually 4–6 l) was 0.4–0.6. The temperature cm in the reduced/carbon monoxide-bound minus re-
was then decreased to 18 8C and the culture allowed to duced absorption difference spectrum. Due to the fact that
equilibrate. The culture was then induced with 100 mM CYP121 is not completely converted to a ferrous-carbon
IPTG and culture continued for a further 20–24 h. After monoxide complex with an A near 450 nm, themax
this time, cells were harvested by centrifugation (75003g, concentration was more accurately determined from the
30 min, 4 8C), washed by resuspension in ice-cold 50 mM Soret maximum of the oxidised enzyme in the resting
Tris–HCl (pH 7.2) plus 1 mM EDTA (buffer A), pooled (almost completely low-spin) state (A 5416.5 nm),max
4. 530 K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541
21 21
using the extinction coefficient ´ 595 mM cm , as relevant concentration of inhibitor. For tight-binding azole416
described previously [22]. inhibitors, data were fitted to a quadratic function, which
accounts for the quantity of the azole drug consumed in
2.4. Mass spectrometry complex with the P450 in determining the K value for thed
inhibitor. The relevant equation is A 5 ((A /(2 3 E)) 3max
2 0.5
Electrospray mass spectrometry was carried out on a (L 1 E 1 K )) 2 ((L 1 E 1 K ) 2 (4 3 L 3 E)) ; whered d
micromass platform quadruple mass spectrometer equipped A represents the observed absorption difference at each
with an electrospray ion source. The cone voltage was set azole addition, A is the maximal absorption differencemax
to 70 V and source temperature to 65 8C. A Waters 2690 at azole saturation, E is the total enzyme concentration and
HPLC unit with a Waters 486 tuneable absorbance detector L is the ligand concentration used. Otherwise, data for
connected to the mass spectrometer was used. Protein was weaker binding inhibitors were fitted to a rectangular
prepared for mass spectrometry by exchange into 0.1% hyperbola. All data fitting was performed using Origin
formic acid through successive ultrafiltration steps using software (Microcal). All titrations were done twice and the
Centricon centrifugal filtration devices (30-kDa cut off, K values reported are the mean for the two sets of data.d
Millipore). Protein (80 mM) was separated on a Jupiter 5 Ketoconazole was also titrated against the P450 in the
mg C-4 300A column at constant TFA concentration presence of a 10 mM concentration of the potential
(0.01%), using a linear gradient of 10–100% acetonitrile in substrate-like molecule lanosterol. Stocks of these com-
21
water over 40 min at a flow rate of 0.05 ml min . Total pounds were prepared in DMSO. In order to minimise
ion count in the m/z range 500–2000 and the UV protein aggregation during the titration in the presence of
chromatogram at 280 nm were recorded for the reverse lanosterol, glycerol was introduced to the assay buffer at a
phase HPLC separation. The mass spectrometer scanned at final concentration of 10%. The apparent K was de-d
0.1-s intervals, the scans were accumulated and the aver- termined as above.
age molecular mass was determined using the MaxEnt and
Transform algorithms of MassLynx software. 2.5.2. Circular dichroism
Circular dichroism (CD) spectra were recorded at 25 8C
2.5. Spectroscopic characterization on a JASCO J600 spectrapolarimeter (calibrated with
0.06% d-10 camphorsulfonic acid). Far UV CD spectra
2.5.1. Electronic spectroscopy were recorded over the wavelength range 190–260 nm in a
UV–visible absorption spectra were recorded on quartz cylindrical cell of 0.02-cm pathlength with a scan
21Shimadzu 2101 and Cary UV-50 Bio UV–visible scanning rate of 10 nm min . Near UV and visible CD spectra were
spectrophotometers using 1-cm pathlength quartz cells. recorded over the wavelength ranges 260–320 nm and
Unless otherwise stated, spectra were recorded using 320–600 nm, respectively, in cells of 0.5-cm pathlength
21approximately 5–10 mM CYP121 enzyme in 50 mM with scan rates of 20 nm min . Spectra were recorded in
Tris–HCl (pH 7.2). Spectra for reduced enzyme were duplicate and averaged. Protein concentrations used were 2
recorded after addition of a few grains of sodium dithion- mM (far UV) and 20 mM (near UV–visible). Secondary
ite. The carbon monoxide complex of CYP121 was structure content analysis was performed using the CON-
generated by slow bubbling of a dithionite-reduced enzyme TIN and SELCON programs [23].
with the gas (for 1 min). Nitric oxide complexes of
CYP121 were obtained by brief bubbling of the buffered
2.5.3. EPR and MCDenzyme solution with nitric oxide gas (approx. five small
Electron paramagnetic resonance (EPR) spectra werebubbles of NO gas released into the solution). Binding of
recorded on an X-band ER-200B spectrometer (Brukerazole inhibitors to CYP121 was measured at 30 8C, usually
Spectrospin) interfaced to an ESP1600 computer and fittedusing 1 mM enzyme. The P450 inhibitors clotrimazole,
with a liquid helium flow-cryostat (ESR-9, Oxford Instru-econazole, ketoconazole, fluconazole and miconazole were
ments). Spectra were recorded at 10 K with 2 mWprepared as stock solutions (typically 0.1–25 mM) in
microwave power and a modulation amplitude of 1 mT.DMSO. Small aliquots (0.1–0.4 ml) corresponding to final
Magnetic circular dichroism (MCD) spectra were recordedconcentrations of 0.01–100 mM of inhibitor were added to
on circular dichrographs, JASCO J-500D and JASCO J-a protein solution, with the total addition less than 10 ml
730 for the ranges 280–1000 nm and 800–2000 nm,inhibitor. Spectra were recorded between 300 to 750 nm
respectively. Samples were mounted in the ambient-tem-after each addition of substrate. Difference spectra were
perature bore of an SM-1, 6 Tesla superconductinggenerated by the subtraction of the original inhibitor-free
solenoid (Oxford Instruments). Protein samples (176 mM)spectrum from each inhibitor-bound spectrum. Binding
were in 50 mM Tris–HCl (pH 7.2).coefficients (K values, often referred to as K values ford s
spectral binding titrations of P450 enzymes) were de-
termined by plotting the maximal absorbance changes 2.5.4. Fluorescence
calculated from each difference spectrum against the Protein (i.e. aromatic amino acid) fluorescence spectra
5. K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541 531
were recorded at 25 8C on a Perkin Elmer LS50B lumines- tions were made from selected transformants, and the clone
cence spectrometer. Measurements were made in the scan verified by restriction enzyme digestion (using NdeI and
mode, using a 1-cm pathlength quartz fluorescence cuvette BamHI restriction enzymes, the sites for which were
and excitation/emission slit widths of 3 nm. Excitation incorporated at the 59 ends of the forward and reverse
was at 290 nm, and emission spectra were recorded primers). The DNA sequence was determined for success-
between 300 and 400 nm. Each measurement was achieved ful CYP121 clones, and found not to deviate from that
by averaging four individual fluorescence scans. Samples reported [2]. The CYP121 gene was then excised from
were in a total volume of 0.5 ml containing 20 mM P450 pGEM-T clone (pKM2a) using NdeI and BamHI, and
enzyme. ligated into the expression vector pET11a, pre-cut with the
same enzymes. Clones of the expression plasmid (pKM2b)
2.5.5. Resonance Raman were verified as before.
A Resonance Raman spectrum was obtained using 15
mW, 406.7 nm radiation at the sample, from a Coherent 3.2. Expression and purification of CYP121
Innova 300 krypton ion laser, and acquired using a
Renishaw micro-Raman system 1000 spectrometer. The Preliminary expression trials indicated that CYP121
sample was held in a capillary under the microscope at a could be expressed to very high levels under control of the
concentration of 50 mM and an extended scan was T7 RNA polymerase promoter system in pKM2b, using a
21
obtained from 200 to 1700 cm , with 12315-s expo- variety of E. coli strains under ‘regular’ growth conditions
sures. (i.e. culture at ca. 37 8C, 250 rpm agitation and induction
with 1 mM IPTG in late log phase). Analysis by SDS–
2.5.6. Materials PAGE showed that the heterologously expressed P450 was
Oligonucleotide primers for PCR were obtained from produced at higher levels than any of the host proteins (at
Perkin Elmer Applied Biosystems (Warrington, UK). All least 25 mg enzyme/l of cells), and cell pellets of the
restriction enzymes and DNA modifying enzymes were induced E. coli transformants were markedly red in colour,
from New England Biolabs (Hitchin, UK). Other modify- indicating high levels of production of the cytochrome
ing enzymes, Taq and Pfu DNA polymerase, and T4 DNA P450 enzyme. However, subsequent breakage of such cells
ligase were obtained from Promega (Southampton, UK). revealed that the bulk of the P450 (.90%) was located in
Unless otherwise stated, all reagents used were obtained inclusion bodies. Solubilization of these using denaturant
from Sigma (Poole, UK) and were of the highest grade (guanidinium chloride) or Bugbuster protein extraction
available. Media and most solutions were made according reagent (Novagen) produced exclusively the inactive
to standard recipes [20]. Econazole, ketoconazole and (P420—see below) form of CYP121. In order to optimise
fluconazole drugs were from ICN (Basingstoke, UK). expression of the native soluble form of CYP121, a variety
Clotrimazole, miconazole and other azoles were from of other growth conditions and host strains were investi-
Sigma. gated. Inclusion of small amounts of detergent in growth
and lysis buffers (e.g. 0.1% Tween 20) was without
beneficial effect on soluble protein recovery. However, low
3. Results growth temperature and mild induction were found to be
essential to promote overproduction of soluble CYP121.
3.1. Cloning of CYP121 Optimal soluble CYP121 production was obtained in E.
coli strain HMS174 (DE3), with IPTG induction (100
The Rv2276 gene (hereafter referred to as CYP121 ) was mM) in the mid-logarithmic phase of growth, and with cell
amplified by PCR using Taq DNA polymerase and for- culture continued at low temperature (18 8C) for approxi-
ward and reverse primers designed from the genome mately 24 h post-induction. Under these conditions, the
sequence. The PCR fragment containing the Rv2276 gene overall expression of CYP121 was lower, but the recovery
was analysed by agarose gel electrophoresis, and verified of soluble CYP121 protein much higher.
to be of the expected size (1.188 kb) by comparison with Typically, CYP121 was purified to homogeneity using
DNA fragment standards (1-kb ladder, Gibco–BRL, UK). ammonium sulfate fractionation followed by column chro-
The fact that Taq DNA polymerase leaves single adeno- matography using Phenyl Sepharose, Q-Sepharose and
sine nucleotide overhangs at the 39 ends was exploited Hydroxyapatite. A final FPLC step (again using Q-Sepha-
through cloning the fragment into the pre-cut pGEM-T rose) was introduced where necessary to obtain ultra-pure
vector, which has single thymidine nucleotide overhangs at P450 protein for crystallization trials. The relative purity of
its 59 ends. Successful clones were identified (following CYP121 was measured during purification by comparing
transformation of the ligation mixture into E. coli strain the heme-specific absorption (at 416.5 nm) with the total
TG1) by the blue/white colony colour selection method protein absorption at 280 nm at different stages of purifica-
[24] on LB agar media containing ampicillin (100 mg/ml), tion. These measurements typically showed that soluble
X-gal (50 mg/ml) and IPTG (50 mg/ml). Plasmid prepara- CYP121 was purified approximately 50-fold from the
6. 532 K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541
Spectrometry (ESIMS) of the purified CYP121 indicated a
single, intact species of molecular mass (M )543 128 Dar
(not shown). This correlates almost exactly with the
predicted mass of CYP121 based on translation of its gene
sequence, once the mass of the initiator methionine has
been subtracted (43 126 Da).
3.3. Spectrophotometric characterization
UV–visible absorption spectroscopy provides the pri-
mary technique for recognition and characterization of
cytochrome P450 enzymes. The oxidized form of pure
CYP121 shows spectral properties typical for members of
the P450 enzyme class, with the major (Soret or g) band
located at 416.5 nm, and the smaller a and b bands at 565
and 538 nm, respectively. On reduction of the pure enzymeFig. 2. Purification of M. tuberculosis CYP121. The SDS–PAGE gel
with sodium dithionite, the Soret band shifts to 405 nmshown (10% acrylamide) indicates the purification of CYP121 from E.
and diminishes in intensity (Fig. 3). These features arecoli HMS174(DE3)/pKM2b cells. Lane 1: SeeBlue2 protein molecular
weight standards (Invitrogen). Mass (from top to bottom, kDa): 250, 148, typical of P450s, and similar to the well-characterized
98, 64, 50 and 36; Lane 2: HMS174(DE3)/pKM2b cell extract; Lane 3: P450 cam (CYP101) and P450 BM3 (CYP102A1) systems
70% ammonium sulfate fraction; Lane 4: post-Phenyl Sepharose; Lane 5:
[25,26], although the spectral maxima for both oxidisedpost-Q-Sepharose; Lane 6: post-Hydroxyapatite (showing 1 mg pure
and reduced forms are both at slightly shorter wavelengthsCYP121 protein at |43 kDa). Lane 7: protein molecular weight standards
(by approx. 2 nm) for CYP121 than for P450s cam and(NEB) (from top to bottom, kDa): 175, 83, 62, 48, 33, 25.
BM3. Under aerobic conditions, CYP121 proved difficult
to reduce completely to the ferrous form. This was due
original cell extract (i.e. CYP121 comprised approximately mainly to the relatively fast re-oxidation of the heme iron
2% of the initial protein content), and was recovered in (compared to the rate of reduction of the ferric heme by
yields of approximately 2–4 mg pure P450 per l cell dithionite). Addition of large excesses of dithionite also
culture. The ratio of heme-specific to total protein absorp- promoted aggregation and precipitation of CYP121, and
tion (i.e. A ) gives a measure of purity, with a value bubbling of carbon monoxide through the dithionite-treated416.5 / 280
of approx. 1.9 indicating homogeneous CYP121, as ver- CYP121 invariably generated a mixture of spectral species
ified by SDS–PAGE (Fig. 2). Electrospray Ionization Mass with absorption maxima at 448 and 420 nm (Fig. 3, inset).
Fig. 3. Electronic absorption spectra for CYP121. The UV–visible absorption spectrum for pure CYP121 (ca. 5.5 mM) in the oxidised (thick solid line),
dithionite-reduced (dashed line), nitric oxide-bound (dotted line) and cyanide-bound (thin solid line) are shown. Soret absorption maxima are located at
416.5, 405, 437 and 438 nm, respectively. The inset shows a difference spectrum generated by subtraction of the spectrum for ferrous CYP121 from the
ferrous-carbon monoxy form. The presence of both P420 (peak near 420 nm) and P450 (peak near 450 nm) forms is evident.
7. K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541 533
The former is indicative of native enzyme (the P450 reveals a mainly alpha helical structure, as expected for a
species), but the latter (P420) indicates that a proportion of cytochrome P450 enzyme. All P450 structures solved to
the enzyme has lost cysteinate ligation following dithionite date are predominantly alpha helical (e.g. Refs. [28,29]).
reduction and exposure to the gas [27]. The fact that this Fig. 4a shows the far UV CD spectrum of Mtb CYP121,
phenomenon is not related to CYP121 in its resting form is compared with that of the heme domain of flavocytoch-
reinforced by the fact that the ratio of P450/P420 varies rome P450 BM3 at the same concentration (3 mM).
between experiments with CYP121 samples from the same Clearly the far UV CD spectra for CYP121 and P450 BM3
batch. Also, the ligation of other inhibitor molecules to the are highly similar. Both the CONTIN and SELCON
ferric P450 results in conversion to a single spectral prediction programs suggest greater than 50% alpha helical
species. For instance, addition of nitric oxide to the ferric content for both the P450s [23]. However, in the near
CYP121 produces a nitrosyl adduct with spectral maxi- UV–visible spectral regions (260–600 nm), CYP121
mum at 437 nm, and ligation of cyanide induces complete shows marked differences from the CD spectra reported
conversion to a species with Soret maximum at 438 nm. In for other P450s in this region (e.g. Refs. [30,31]). In the
addition, our recent determination of the atomic structure near UV region (260–320 nm), signals arise mainly from
of CYP121 confirms that the protein is cysteinate-ligated aromatic amino acid side chains, and CD spectra in this
(D. Leys et al., in preparation). area are characteristic of individual P450 isoforms. In the
In binding trials with a variety of fatty acid, steroid and visible region (320–600 nm), the CD spectra of P450s
sterol and polyketide compounds, it was found that the characterized to date are dominated by a large signal of
predominantly low-spin spectrum of CYP121 (A at negative sign, with a minimum near the position of themax
416.5 nm) was not perturbed to any significant extent. Soret maximum in the electronic absorption spectrum.
Most P450s show a shift in heme spectrum on binding However, for CYP121 the Soret visible CD band is clearly
their substrates, due to perturbation of the heme iron of positive sign, and of similar intensity as the negative
spin-state equilibrium in favour of the high-spin form. This band for the P450 BM3 heme domain (Fig. 4b). For
results in a shift of the Soret band maximum from close to CYP121, the visible CD maximum is at 419 nm, close to
420 nm (low-spin) to approximately 390 nm (high-spin). the maximum in the electronic absorption spectrum (416.5
The fact that no such perturbations were induced with nm). The reason for this unusual CD spectrum is unclear,
CYP121 suggests that the molecules tested thus far do not but may relate to a slightly ‘kinked’ heme conformation
mimic closely the structure of the true CYP121 sub- seen in the recently solved atomic structure of CYP121 (D.
strate(s). Our recent structural determination of CYP121 Leys et al., in preparation).
indicates it has a very large active site cavity, perhaps
reflecting that the natural substrate is a bulky and complex 3.4.2. Electron paramagnetic resonance
lipid. However, it should also be noted that rather minor The X-band Electron Paramagnetic Resonance (EPR)
shifts in the spin-state equilibrium are noted for the Mtb spectrum for oxidised (ferric) CYP121 is shown in Fig. 5.
sterol demethylase CYP51 on binding the sterol ob- The major signals in the spectrum are a rhombic trio of
tusifoliol [12], suggesting that any substrate-dependent g-tensor elements (at g52.47, 2.25 and 1.90), typical for a
spectral perturbation observed for Mtb CYP121 may also low-spin ferric heme iron. Other minor signals in the
be minor. spectrum at g54.29 and 5.9 are due to adventitious Fe(III)
In the P450 cam system, elevation of temperature has and high-spin ferric heme iron, respectively. The sharp,
also been shown to perturb the heme iron spin-state narrow signal at approx. g52 is due to a minor radical
equilibrium in favour of the high-spin form [25]. However, contaminant, at much lower concentration than the P450.
elevation of the incubation temperature of a buffered The CYP121 EPR spectrum is virtually identical to those
solution of CYP121 (5 mM) in the temperature range reported previously for the well-characterized P450 cam
between 20 and 45 8C caused only very minor changes in (g52.46, 2.26 and 1.91) and P450 BM3 (2.42, 2.26 and
the CYP121 Soret spectrum, indicating that the heme 1.92) [32,26]. The values are also very similar to those
remains predominantly low-spin in this temperature range. reported for different isoforms of nitric oxide synthase
Elevation of the temperature to 40 8C resulted in aggrega- (NOS), which also have cysteinate- and water-ligated
tion and precipitation of CYP121, and although inclusion heme iron (e.g. Refs. [33,34]).
of glycerol (10% v/v) prevented aggregation until approx.
45 8C, there was still little spin-state perturbation observed. 3.4.3. Magnetic circular dichroism
Evidently the CYP121 protein stabilizes strongly the low- The UV–visible and near IR Magnetic Circular Dichro-
spin form of the heme. ism (MCD) spectra for oxidised CYP121 are shown in Fig.
6. MCD spectra in the UV–visible region are particularly
3.4. Spectroscopic and biophysical characterization diagnostic of the spin- and oxidation-states of metal ions,
and in hemoproteins the optical bands from the porphyrin
3.4.1. Circular dichroism macrocycle are sensitive to the properties of the central
The far UV (190–260 nm) CD spectrum of CYP121 iron. Features at wavelengths between 300 and 600 nm are
8. 534 K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541
Fig. 4. CD spectra for CYP121. (a) An overlay of far UV CD spectra (190–260 nm) for CYP121 (solid line) and the heme domain of flavocytochrome
P450 BM3 (both 3 mM). (b) An overlay of CYP121 (solid line, 20 mM) and BM3 heme domain (dotted line, 20 mM) in the near UV–visible region. All
spectra were recorded at 20 8C in 50 mM Tris–HCl (pH 7.5) as described in the Experimental section.
due to p-to-p* transitions in the porphyrin ring. Mixing of peak at approx. 290 nm is due to tryptophan residues in
porphyrin-p with iron-d electronic levels occurs to an CYP121. As with the EPR spectra, the visible MCD
extent so that the UV–visible MCD spectra are informative signals for CYP121 are highly similar to those observed
of spin- and oxidation-state of the iron. As described previously for other bacterial P450s [32,35–37] and for a
above, the electronic absorption spectrum of ferric liver microsomal P450 [37]. There is a small trough in the
CYP121 has characteristic bands at 416.5, 565 and 538 nm MCD at |655 nm corresponding to the weak broad
(Fig. 6, panel A). The major features in the UV–visible absorption observed at 620–680 nm. This is likely due to a
MCD spectrum of the same sample (Fig. 6, panel B) are weak charge transfer (CT) band arising from a small
typical in wavelength and shape for low-spin ferric hemes. population (|5%) of high-spin thiolate-coordinated ferric
21 21
However, the relatively low intensities (15.5 M cm heme.
21 21 21
T peak to trough in the Soret band and 23.9 M cm Cytochromes containing low-spin ferric heme give rise
21
T for the a-band trough at 575 nm) are unique to to a porphyrin (p)-to-Fe(III) CT transition that appears as a
low-spin hemes with thiolate ligation. The sharp positive weak positive band in the near IR region. The exact energy
9. K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541 535
21
CYP121 these are located at 1487 and 1500 cm , for
high-spin and low-spin ferric heme, respectively. The
intensity of the low-spin band is much greater. In the case
of P450 BioI, the situation is reversed [19]. The overall
resonance Raman spectrum of CYP121 is typical of a
native cysteinate-ligated P450 enzyme, with negligible
P420 content.
3.5. Structural stability of CYP121
Due to the apparent instability of CYP121 to treatments
such as reduction/bubbling and heating, we examined
effects of the denaturant guanidinium chloride (GdmCl) on
the structural integrity of the enzyme and its heme
cofactor. UV–visible absorption, CD and fluorescence
spectra were recorded for CYP121 incubated with GdmCl
in the concentration range between 0 and 6 M, in order to
assess the effects of the denaturant on secondary and
tertiary structure, and on heme binding. In the visibleFig. 5. EPR spectrum for CYP121. The X-band EPR spectrum of
absorption spectrum, concentrations of GdmCl as low asCYP121 (176 mM) is shown. The spectrum was recorded at 10 K in 50
0.2 M induced decreases in the intensity of the hememM Tris–HCl (pH 7.5), as described in the Experimental section.
spectrum in the Soret region. There was an approximately
50% decrease in Soret intensity at 0.5 M GdmCl, sug-
of this band varies systematically according to changes in gesting that either considerable amounts of heme are lost
the axial ligation state, and is thus diagnostic of the nature from the protein under these conditions, or else the
of the heme ligation for the particular cytochrome investi- CYP121 heme structure is disrupted. Considerable turbidi-
gated [35]. For CYP121 (Fig. 6, panel 3), this weak band ty of the CYP121 protein solution was evident in the range
is located at approximately 1125 nm. This is similar to the between 0.75 and 1 M GdmCl, indicating protein aggrega-
position of this CT band in P450 BM3 (1060 nm) and the tion and precipitation. However, at higher [GdmCl] there
B. subtilis biotin synthesis pathway P450 BioI (1090 nm), was negligible turbidity, showing that more concentrated
suggesting similar cysteinate/water ligation occurs in denaturant solutions kept the unfolded polypeptide in
CYP121 [38]. solution effectively. The Soret CD signal was also seen to
diminish by more than 80% in the GdmCl concentration
3.4.4. Resonance Raman range between 0.5 and 1 M. Evidently, the structure of the
A resonance Raman spectrum of the CYP121 (shown in heme macrocycle is sensitive to quite low concentrations
Fig. 7) was obtained with excitation at 406.7 nm. The of denaturant. With regard to the stability of the secondary
spectrum (which shows strong similarity to that obtained structure of CYP121, far UV CD studies indicate that there
for other P450 enzymes) is labelled according to previous is slightly increased helicity of the protein at 0.5 M
assignments [39–41]. The 406.7-nm excitation is close to GdmCl, a phenomenon that has been observed for a
the Soret transition of the heme chromophore (416.5 nm) number of other proteins [42]. However, CYP121 under-
and gives Raman scattering from the vibrational modes of goes considerable substantial unfolding in the range be-
the porphyrin ring. The dominant oxidation state marker tween 0.5 and 2 M. By fitting the changes in ellipticity at
21
band, n , at 1372 cm , indicates that the heme is in the 220 nm for CYP121 to a rectangular hyperbola, the4
ferric state, in agreement with electronic, EPR and MCD midpoint for the unfolding of the enzyme was found to be
data. The spin state markers, n , n , n , n , n , and n , 1.960.1 M GdmCl. Changes in tryptophan fluorescence3 11 2 37 38 10
21
between 1480 and 1650 cm are sensitive indicators of were used to monitor stability of the tertiary structure
the heme core size, and thus the spin-state of the heme under the same conditions. With excitation at 290 nm, an
iron. These reveal that there is a proportion of high-spin approximately threefold increase in tryptophan fluores-
ferric heme iron along with the predominant low-spin cence was noted between the fully folded enzyme (0 M
form. While we cannot rule out the possibility that heating GdmCl, l emission5347 nm) and the fully unfoldedmax
by laser irradiation causes a small increase in the high-spin form (6 M GdmCl, l emission5356 nm). In accord-max
content of CYP121, the proportion of the high-spin form is ance with the results of far UV CD, there was a negligible
much greater, for instance, in the B. subtilis P450 BioI fluorescence increase in the range between 0 and 0.5 M
enzyme, which is isolated in a mixed spin-state form [19]. GdmCl. However, fluorescence changes indicated progres-
This is most clearly evident from the nature of the splitting sive loss of tertiary structure above 0.5 M, with 50% of the
of the n band into two well-resolved components. In total change in tertiary structure occurring at 1.260.1 M3
10. 536 K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541
Fig. 6. MCD spectra for CYP121. The UV–visible absorption spectrum for CYP121 (panel A) is compared with the MCD spectrum in the same region
(panel B). Panel C shows the MCD spectrum for CYP121 in the near IR region (units same as in panel B). Protein concentration in all cases is 176 mM.
MCD spectra were recorded at room temperature as described in the Experimental section.
GdmCl. While these data indicate that CYP121 is not a too tight to analyse accurately, even at low concentrations
particularly robust enzyme, comparable studies for the of CYP121 (1 mM). For these three azoles, the optical
GdmCl-induced unfolding of Mtb CYP51 indicate this change associated with azole ligation (shift of the Soret
putative sterol demethylase to exhibit rather lower structur- maximum to approximately 424 nm) occurs linearly with
al stability. In the case of CYP51, far UV CD studies azole concentration, sharply reaching a plateau and indica-
indicate that the protein is 50% unfolded by only 0.65 M tive of stoichiometric binding to the P450. Fitting to the
GdmCl. described quadratic equation does not provide satisfactory
solutions, due to the tight binding. Evidently the K valuesd
3.6. Azole drug binding properties for these azoles are very low, i.e. ,0.2 mM. Optical
titrations with fluconazole revealed rather weaker binding,
In view of the fact that azole antifungal drugs (known with a K of 9.760.1 mM, determined from a hyperbolicd
sterol-metabolizing P450 inhibitors) have been shown to fit of the absorption change versus fluconazole concen-
bind efficiently to the Mtb CYP51 [11–13], we tested tration. Ketoconazole bound to CYP121 with slightly
CYP121 to determine its affinity for members of this class higher affinity (K 53.360.3 mM). A repeat of thed
of drugs. The azole antifungal agents clotrimazole, ketoconazole binding titration in the presence of lanosterol
econazole, fluconazole, ketoconazole and miconazole were (10 mM) showed that affinity for the azole was increased
found to bind very tightly to CYP121. Binding of clot- considerably (K 50.360.05 mM, Fig. 8), indicating thatd
rimazole, econazole and miconazole to CYP121 proved lanosterol promotes the binding of ketoconazole to
11. K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541 537
21
Fig. 7. The resonance Raman spectrum of CYP121 (50 mM) was recorded between 1700 and 200 cm at room temperature with excitation at 406.7 nm,
and 12315-s exposures for data collection, as described in the Experimental section. Positions of selected, assigned vibrational bands are shown, including
21
the n marker band for oxidised ferric heme (1372 cm ), and the split n feature indicating the presence of a small amount of high-spin heme (HS, 14874 3
21 21
cm ) alongside the predominant low-spin component (1500 cm ).
CYP121, and that the active site of the enzyme can obvious of the Mtb CYP enzymes for characterization was
accommodate both the azole and the sterol. The structure the CYP51 encoded by the Rv0764c gene. This was the
of CYP121 confirms that the substrate-binding cavity of first recognized prokaryotic homologue of sterol de-
the P450 is significantly larger than those of most other methylase P450s found in eukaryotes [43,44]. The yeast
bacterial P450s [D. Leys et al., in preparation]. Interesting- and fungal CYP51 enzymes are lansoterol demethylases
ly, reduction of azole antifungal-bound forms of CYP121 involved in synthesis of the essential membrane sterol
with dithionite generated a distinct spectral form with ergosterol [45]. Inhibition of ergosterol synthesis by azole
Soret maximum at 427 nm, an alpha band of increased drugs (e.g. ketoconazole, fluconazole etc.) disrupts mem-
intensity at 559 nm, and a weaker beta band at 531 nm. brane integrity and is an effective antifungal therapy [15].
Thus the spectral changes induced on reduction of the Thus, the possibility that a similar strategy could provide
CYP121 azole complexes are similar to those observed for an attractive new antibiotic therapy for Mtb was raised.
the ferrous forms of sulfur/histidine ligated cytochromes Expression of the Mtb CYP51 and demonstration of azole
(e.g. cytochrome c), as might be expected for the reduced drug-binding has been reported by various groups [11–13].
form of a cytochrome with axial azole and cysteinate Moreover, the atomic structure of the Mtb CYP51 was also
ligands. solved in the presence of both fluconazole and the smaller
azole compound 4-phenylimidazole [45]. The structures
reveal a large active site cavity and an unusual ‘kinked’
4. Discussion conformation of the I alpha helix of the P450 (which
contains several residues critical for structure and catalytic
The large numbers of cytochrome P450-encoding (CYP) properties). There are also several stabilizing interactions
genes in the Mtb genome indicate that there are essential between protein side chains and the aromatic rings of the
roles for these enzymes in the physiology of the pathogen. inhibitors, as well as the ligation of the azole groups of the
However, little is currently known about the enzymology inhibitors to the ferric heme iron. Mtb CYP51 binding to a
or biophysical features of these enzymes. The most variety of azole antifungal drugs is relatively tight (Kd
12. 538 K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541
Fig. 8. Azole drug binding to CYP121. Difference spectra resulting from the titration of CYP121 (6.5 mM) with ketoconazole in the concentration range
between 0 and 25 mM are shown in the main figure. In the absolute spectra for the oxidised CYP121, ketoconazole saturation results in the shift of the
heme Soret band from 416.5 to 424 nm. Subtraction of the starting (azole-free) spectrum from that of each azole-bound form (at 0.2, 1.2, 2.2, 3.2, 5.2, 7.2,
13.2 and 25 mM) generates the series of difference spectra shown, with progressive deviation from the baseline observed with increasing [ketoconazole].
The maxima and minima in the difference spectra are located at 430 and 396 nm, respectively. The inset shows ketoconazole-induced absorption change
versus [ketoconazole] data for CYP121 spectral titrations performed as described (black circles) and in the presence of 10 mM lanosterol (black triangles).
Data points are fitted to the quadratic expression cited in the Experimental section, yielding K values of 3.360.3 mM for ketoconazole alone, andd
0.360.05 mM in the presence of lanosterol. Similar differences were obtained at lower CYP121 concentrations.
values in the mM range), raising the possibility that the ly both polyketide- and sterol-metabolizing P450s can
CYP51 could be a new anti-Mtb drug target. Novel drugs exhibit high affinity for these azoles.
targets are desperately needed due to the prevalence of Preliminary expression studies with CYP121 demon-
drug-resistant strains of the pathogen. Preliminary work strated that it could be produced at high levels in E. coli,
suggests that the azoles are effective in preventing growth but that strong induction resulted in the bulk of CYP121
of mycobacteria [11,13], but CYP51 remains non-validated forming inclusion bodies. This problem was overcome by
as the true target for the drugs. Indeed, with such a slowing bacterial growth (low growth temperature) and
plethora of P450s encoded by Mtb, there are potentially a decreasing IPTG inducer concentration. While the problem
number of target P450s. with expression of soluble CYP121 may in part be related
In this paper, we report the expression and characteriza- to the relatively high GC-content of the Mtb CYP121 gene
tion of a second Mtb P450–CYP121. This P450 also (62.2% GC), it may also be the case that the CYP121
exhibits high affinity for azole antifungal drugs. Indeed, enzyme is naturally weakly associated with the Mtb
the affinity for commercially available azole drugs is membrane. Certainly, there are long stretches of hydro-
higher than that reported for Mtb CYP51 [12,13]. Clearly phobic amino acid sequence in the P450 that might
CYP121 is also a potential azole drug target in Mtb. While mediate such interactions (e.g. between T36 and L55, and
work is ongoing to establish its biological function and between L222 and I250), and the ease with which the
substrate selectivity, the fact that it binds even tighter to enzyme aggregates and precipitates on heating and reduc-
azole drugs than does CYP51 is an exciting finding as tion/bubbling suggests that the phenomenon may be
regards the possibility that drugs of this class may have mediated by interactions between such hydrophobic re-
more than one target in Mtb. Should this drug strategy gions on the CYP121 surface. The high affinity for
prove effective against Mtb, then the presence of multiple hydrophobic, polycyclic azoles, coupled to the fact that the
targets should minimise the possibility of development of presence of lanosterol can promote the binding of
drug resistance. Previous studies on the polyketide- ketoconazole, suggests that the true substrate for CYP121
metabolizing P450 eryF (one of the most highly related should be a complex, lipophilic compound, possibly a
P450s to CYP121 as regards primary structure) demon- polyketide or sterol-like molecule. Such molecules should
strate that it also has high affinity for an azole drug be mainly membrane-located, and association with the Mtb
(ketoconazole, K |3 mM), and the P450 eryF structure has membrane would provide best access for CYP121 to thesed
been solved in the ketoconazole-bound form [46]. Evident- potential substrates.
13. K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541 539
Biophysical studies of the CYP121 enzyme indicate that synthesis cluster, and a P450 from Actinomadura hibisca
it exhibits many properties that are typical of the P450 involved in pradimicin biosynthesis [17,48,49]. Elsewhere
class. As shown, by electronic absorption, resonance in the amino acid sequence of CYP121, residues recog-
Raman, EPR and MCD spectroscopy, CYP121 is pre- nized as an important heme binding motif are retained
dominantly low-spin in its resting oxidized state. There is a (including the cysteinate heme ligand [C345] and a
small high-spin component (,5%), as evident from the phenylalanine important for control of redox properties of
resonance Raman spectrum (splitting of n3 component, the heme iron [F338]) [50] (Fig. 1). A serine residue
Fig. 7), EPR studies (Fig. 5), and from the minor ab- (S236) is located at the same position as a threonine
sorbance shoulder near 390 nm in the electronic absorption known to be important for oxygen binding/activation and
spectrum (Fig. 3). There is strong stabilization of the proton transfer in P450 cam (T252) and P450 BM3 (T268)
low-spin form, as evident from the minimal effect of [50,51]. However, approximately 10% of other P450s also
heating CYP121 on the spin-state equilibrium. EPR, MCD contain the alternative hydroxyl side chain amino acid
(Fig. 6) and resonance Raman all indicate that CYP121 serine at this position. In the case of P450 eryF, an alanine
has typical P450 cysteinate- and water-ligation to the heme (A245) is present instead, but in this enzyme the substrate
iron, and there is negligible P420 content. Thus, the fact itself (6-deoxyerythronolide B) participates in dioxygen
that a considerable amount of P420 forms during bubbling cleavage [18]. Amino acids involved in ketoconazole
of dithionite-reduced CYP121 with carbon monoxide (Fig. binding to P450 eryF and their corresponding residues in
1, inset) results from the sensitivity of the enzyme to CYP121 are highlighted in grey in Fig. 1. In the P450
reduction and/or agitation required to form the carbon eryF-ketoconazole complex, the majority of interactions
monoxide adduct (i.e. is an artefact of the preparation with the drug come from hydrophobic side chains, with
process). Once the carbon monoxide complex of CYP121 additional hydrogen bonding interactions from tyrosine 75
is formed, the ratio of P450/P420 remains constant. and asparagine 89. One of the hydrogen bonding residues
However, previous studies of the Mtb CYP51 suggest that is conserved in CYP121 (N84), and lipophilic amino acids
the P450 complex in this enzyme is unstable, with P450 in the I helix region of CYP121 are also conserved (A233)
converting to the inactive P420 within a few minutes of or retained as hydrophobic residues (T229, S237), sug-
formation of the complex [43]. Stability studies of gesting that these residues may also have important roles
CYP121 confirm that it is rather unstable to heating and to in substrate/azole antifungal interactions in CYP121.
treatment with denaturant (GdmCl). However, similar Trials with a range of potential substrate molecules
studies on Mtb CYP51 indicate that this enzyme is even failed to induce a further significant shift in heme iron
less stable than CYP121, and unfolds at a lower con- spin-state equilibrium towards the high-spin form in
centration of GdmCl [31]. CYP121. Typically, substrate-induced spin-state perturba-
Perhaps the most intriguing spectroscopic property of tion is a feature of bacterial P450s (e.g. Ref. [26]). This
CYP121 is its positive CD signal in the visible region (Fig. finding probably indicates that the natural substrate for
4b). Other P450s characterized to date show a signal of CYP121 is a bulky and complex molecule, as suggested by
negative sign in the Soret region of their visible CD the large active site volume of CYP121 (D. Leys et al., in
spectrum. The positive signal from CYP121 may reflect preparation). Only minor shifts in spin-state equilibrium
non-standard electronic features or heme conformation. In are induced in the Mtb CYP51 on binding non-myco-
this respect, the atomic structure of CYP121 indicates that bacterial sterols, and thus we expect that both Mtb
its heme macrocycle is ‘kinked’ at one of the pyrrole rings, CYP121 and CYP51 will exhibit much greater perturba-
due to the effect of Pro 346, immediately following the tions on interaction with the true physiological substrates.
cysteine heme ligand (Fig. 1; D. Leys et al., in prepara- Given that several of the Mtb P450s have highest similari-
tion). A proline in similar position is found in only a ty to others in the same bacterium, it is plausible that they
handful of other P450s: specifically mammalian cholesterol catalyse oxygenation reactions with substrates of similar
7a-hydroxylases and the Agrobacterium tumefaciens P450 chemical structure, and that these substrates may be
pinF1 (CYP103). The CYP103 is inducible by plant peculiar to the genus (or even species). M. tuberculosis is
phenolics (e.g. acetosyringone) and is presumably involved known to have a very complex lipid metabolism, and much
in oxygenation of such molecules [47]. There is obviously remains to be learned about the nature of many of the
the possibility that the presence of an unusual proline lipids in this organism [9].
residue following the cysteine ligand could relate to P450 In future work, we intend to identify the natural
substrate selectivity for complex polycyclic molecules. substrate by performing binding studies with CYP121
Among the non-mycobacterial P450s most closely related using extracts from Mtb and/or from infected human cells.
to CYP121 in amino acid sequence, many are known (or We are also investigating the redox partners for CYP121
suspected) to have roles in polyketide synthesis; including and the other Mtb P450 systems. To this end, we have
CYP105A1 from S. griseus (which also binds polycyclic recently expressed and purified the ferredoxin reductase-
compounds and is involved in herbicide degradation), a like FprA (product of the Rv3106 gene), and are also
P450 from Streptomyces lavendulae in the mitomycin characterizing potential ferredoxin-encoding genes from
14. 540 K.J. McLean et al. / Journal of Inorganic Biochemistry 91 (2002) 527–541
P.R. Wheeler, N. Honore, T. Garnier, C. Churcher, D. Harris, K.Mtb. Our long-term aim is to define structure and selectivi-
Mungall, D. Basham, D. Brown, T. Chillingworth, R. Connor, R.M.
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