1. Characterization of Radical S-adenosyl-L-methionine
Epimerase, NeoN
Samender Randhawa1, John Zhang1, Yuan Xia1, Cindy Hunt1, Daniel P. Dowling1
1Department of Chemistry, University of Massachusetts Boston, Boston, MA
Funding for this research was provided by the Oracle Education Foundation grant to the CSM
Abstract
A vast number of enzymes are being characterized that belong to a superfamily
known as radical S-adenosyl-L-methionine (SAM) enzymes, whose members
contain a [4Fe–4S] cluster ligated by three cysteine residues. A subset of
radical SAM enzymes, such as the Neomycin C epimerase (NeoN), contain
additional iron–sulfur clusters that are required for the reactions they catalyze.
The goal of this work is to grow NeoN protein crystals and obtain its
crystallographic information in order to understand the underlying biochemical
basis for its catalytic activity. We hypothesize that NeoN could be used as a
biocatalyst tool in the future for developing specific epimerases that could
produce novel compounds.
Methods and Development
Conclusions
Introduction
Future Work
Radical S-adenosyl-L-methionine (SAM) enzymes (RS enzymes)
carryout a variety of biological functions, such as synthesis of cofactors, and
antibiotics1. Till date 113,6334 RS function units have been characterized both
biochemically and structurally. Majority of the RS enzymes adopt a full or
partial triosephosphate isomerase barrel (TIM) fold, with one of the first
reported RS enzyme structures, that of biotin synthase (BioB), adopting a full
TIM barrel fold2 (Fig.1a). The presence of a [4Fe- 4S] cluster bound to a
conserved cysteine triad (CxxxCxxC) is common to all RS enzymes, and the
cluster is positioned at the carboxy terminus of the protein barrel. SAM binds
to the cluster within the barrel, and the substrate (dethiobiotin for biotin
synthase3) binds proximal to SAM, poised for catalysis (Fig. 1b). The RS
enzymes use a common mechanism: the generation of a primary carbon-
centered radical intermediate, the 5’- deoxyadenosyl radical (dAdo●) (Fig.2).
NeoN is a recently characterized epimerase that plays a vital role in the
last biosynthetic step of neomycin B, an aminoglycoside antibiotic produced
by Streptomyces fradiae1. NeoN is a RS enzyme that selectively epimerizes the
C-5''' carbon of neomycin C1 (Fig.3). We are interested in how NeoN is
capable of specifically catalyzing the epimerization of one site of neomycin C,
and future studies of this system may lead to the development of
bioengineered epimerases.
Aims:
• To obtain atomic resolution information in order to characterize and
understand how NeoN functions.
• To identify important residues within the active site that play a role in
catalytic epimerization.
• To characterize substrate recognition in order to bind other non-substrate
molecules for specific epimerization reactions.
• Cloning of NeoN genome, expressing, and crystallizing the protein.
• Ensuring that the expressed crystallized protein is soluble and functionally
active.
• Analyzing the active site residues to explore incorporating non-substrate
molecules for possible epimerization by the enzyme.
The main idea behind crystallizing a protein is that most proteins are
soluble at physiological conditions, but as the concentration of solutes rises, the
protein becomes less soluble, leading it to crystallize or precipitate. The goal of
crystallization is to produce a well-ordered crystal lattice that is able to provide a
diffraction pattern on exposure to X-rays.
The diffraction pattern can then be analyzed using computational programs
to obtain an electron density map, which reveals the protein’s structure and the
binding of cofactors and ligands. The overall scheme of crystallizing NeoN is
shown in figure 5.
Figure 4. RS enzyme
sequence similarity.
Sequence analysis can be
used to assign function to
proteins. A similarity cut
off e-value of 10-28 has
separated the RS enzyme
sequences into clusters
that largely group by
reaction type.
• Epimerization of Neomycin C to Neomycin B is accomplished by NeoN
through its two [4Fe-4S] clusters at C-5’’’.
• A thorough understanding of the NeoN structure for Neomycin B synthesis
will open up the possibility of modifying the enzyme to recognize different
types of substrates, making NeoN a tractable tool as a biocatalyst.
References:
1. Fumitaka Kudo. et al. JACS (2014) 136, 13909-13915.
2. Shisler, Krista. et al. Curr. Opin. Struct. Biol. (2012) 22, 701-710.
3. Berkovitch, F. et al. Science (2004) 303, 76–79.
4. SFLD - Superfamily List." SFLD - Superfamily List. Web. 26 Apr. 2015.
Figure 2. Reductive
cleavage of SAM. RS
enzymes use [4Fe-4S]
clusters to bind to SAM and
transfer an electron to the
sulfonium of SAM,
producing methionine and
dAdo● through homolytic
cleavage of S-5’C bond in
SAM. The dAdo● then
abstracts an H-atom from
the substrate to initiate a
radical-mediated
transformation.
Figure 5. Protein X-ray crystallography methods scheme. We will be cloning
the gene for NeoN from Streptomyces fradiae into an expression vector
containing a hexa-histidine affinity tag for protein purification. Purified protein
will be concentrated and used for crystallization experiments.
a
Figure 3. NeoN epi-
merization of Neomycin C
to Neomycin B. (a) NeoN
is a putative RS enzyme
which is encoded in the
neomycin gene cluster. This
gene cluster is structurally
related to aminoglycoside
biosynthetic gene clusters
such as lividomycin B and
paromomycin1. (b) The first
cluster is made up of C26,
C30, and C33. The second
cluster involves C247,
C249 C271, and C274.
NeoN
C C C C 416C C C
b
b
Figure 1. Structure of BioB. (a) Overall
TIM barrel structure is colored in red (α
helices) and yellow (β strands). (b) The
cofactors and substrates are arranged
vertically as follows: [4Fe-4S] cluster on
the bottom, SAM, dethiobiotin, and the
[2Fe-2S] cluster. Carbon atoms are
colored cyan for substrates. PDB 1R30.
a b