1. Inhibition of pNPG Hydrolysis__
Tight-Binding Transition-State Mimics
• Early inhibition studies tested whether various
sugars could inhibit MalA from cleaving pNPG.
• Despite identifying many compounds that acted
as competitive inhibitors, no new substrates
were found.
• Investigating compounds similar to the
hypothetical transition state can give insight into
the nature of MalA’s catalytic active site and
mechanism of action.
Purification and Characterization of MalA,
an α-Glucosidase from Bdellovibrio bacteriovorus
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Absorptionat280nm(AU)
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Fraction
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Velocity-1(AU/min)-1
[pNPG]-1 (mM)-1
[DNJ] = 8.0 µM
[DNJ] = 4.0 µM
[DNJ] = 2.0 µM
[DNJ] = 0.0 µM
Compound Ki
pNPG 1.5 mM (Km)
Maltose 1.3 - 1.8 mM
Glucose 1.4 - 2.4 mM
DNJ 3.5 µM
Acarbose 1.7 nm
Background__________________
• Bdellovibrio is a predatory gram-negative
bacteria; it prey include E. coli, Staphylococcus,
and other pathogens of human interest.
• Despite years and considerable efforts,
Bdellovibrio and its unique predation
techniques remain poorly understood.
• Genome sequence published in 2005 paved the
way for progress; it contained a gene MalA,
which appeared to code for a maltase-like
enzyme.
• This ongoing collaborative project aims to
better understand Bdellovibrio through the
potentially integral, carbohydrate-cleaving
glycosidase MalA.
MalA________________________
• MalA has only two known substrates: maltose
(and other maltooligosaccharides), and pNPG.
• MalA can bind to a variety of other α-linked
carbohydrates and pseudo-carbohydrates, but
does not interact with them catalytically.
Purification of MalA from E. coli
Cell Prep, Lysis, Filtration
• In 2009 the Martin Lab established a strain of
kan-resistant pMalA E. coli from which
overproduced MalA enzyme can be extracted.
J. Jared Trecker*†, John Hanson†, Mark Martin‡ | †Dept. of Chemistry, ‡Dept. of Biology, University of Puget Sound, Tacoma, WA
Anion Exchange Chromatography
Hydrophobic Interaction Chromatography
TOP10 E. coli
pMalA
= MalA enzyme
LB + Kan
transfer single colony to
establish liquid cultureplate
centrifugation:
collect pellet, resuspend in
phosphate buffer, sonicate
centrifugation:
collect and filter
supernatant
centrifugal filtration:
exchange into chromatography
buffer, concentrate protein
Fig. 3 UV trace recording absorbance changes at 280 nm during
anion-exchange purification of concentrated pMalA E. coli cell lysate.
Lysate in imidazole buffer (10 mM, pH 7.0) was pumped onto a
column packed with diethylaminoethyl groups (DEAE); fractions of 5-
8 mL were collected during elution by a gradient of increasing NaCl
concentration. Superimposed columns represent the enzymatic
activity present in each collected fraction; negligible overlapping of
trace peaks with activity columns suggests significant removal of
contaminating proteins.
Fig. 4 UV trace and activity columns from hydrophobic-interaction
purification of post-DEAE MalA solution in phosphate (50 mM) and
ammonium sulfate (1 M). The solution was pumped onto a column
packed with phenyl groups (pH 7.0), then eluted by a gradient of
decreasing ammonium sulfate concentration. Large UV peaks
recorded after fraction collection (not shown) suggests removal of
many contaminating proteins left after DEAE.
DEAE
Phenyl
Fig. 1 The classical Koshland double displacement mechanism of
hydrolytic cleavage, depicted with generic amino acid residues acting
as acids and bases. Recent NMR experiments have demonstrated
that MalA catalyzes the hydrolysis of maltose (1) to two glucose
molecules (5) according to a stereochemistry-retaining α-glucosidase
mechanism that is common among enzymes in the GH13 family, and
similar to the one here. The enzyme distorts its substrate into a
carbenium-ion-like transition state (3) before water attacks and
completes the second SN2 substitution.
pNPG pNP
Fig. 2 Para-nitrophenol α-D-glucopyranoside (pNPG) is a synthetic
compound that behaves similarly to many α-linked disaccharide
compounds. When cleaved, it forms para-nitropnenol (pNP) and
undergoes a distinct color change that can be easily monitored by
spectrophotometry at 405 nm, and has been used here extensively
to assay MalA’s glucosidase activity.
1
2
3
4
5
Literature Cited
1: Lambert, C.; Chang, C.; Capeness, M.; Sockett, E. The first bite—profiling the predatosome in the bacterial pathogen
Bdellovibrio. PloS one 2010, 5, e8599.
2: Rendulic, S et al. A predator unmasked: life cycle of Bdellovibrio bacteriovorus from a genomic perspective. Science
2004, 303, 689-92
3: Simpson, A. University of Puget Sound Undergraduate Thesis, 2015.
4: Kirkpatrick, G. B.S. University of Puget Sound Undergraduate Thesis, 2010.
5: Gloster, T., Davies, G. Glycosidase inhibition: assessing mimicry of the transition state. Organic & Biomolecular
Chemistry 2010, 8, 305-320.
6: Isabella, C. B.S. University of Puget Sound Undergraduate Thesis, 2012.
7: Smith, P. University of Puget Sound Undergraduate Thesis, 2013.
Acknowledgements
I would like to express my thanks to professor John
Hanson for his support and assistance throughout this
project, to professor Mark Martin for his support and
microbial knowledge, and to the University of Puget Sound
for funding this work.
Fig. 6 Hydrolysis of pNPG by purified MalA in the presence and
absence of dilute deoxynojirimycin. A Km value of 1.5 and a Vmax of
0.163 AU/min was determined for pNPG; a KI value of 3.5 µM was
determined for DNJ.
Fig. 5 Acarbose and dexoynojirimycin (DNJ) are sugar analogs that
act as tight-binding inhibitors of many α-glucosidases. Acarbose has
a planar orientation similar to the dismantled chair of a carbenium-
like transition state, while the nitrogen group in DNJ can a acquire a
positive charge in the same ring location that the transition state
would have a partial dipole; these compound may bind enzymes with
more affinity than their native substrates due to this mimicking.
Table 1 Kinetic parameters measured for various MalA inhibitors.
Acarbose
Deoxynojirimycin
(DNJ)
Deoxynojirimycin
• Having identified acarbose as a potent inhibitor of
MalA in previous studies, DNJ was selected as a
compound that might be similarly potent
Time-Dependent Inhibition
Assessing Possible Isomerization Activity
• Slow-onset inhibition is often observed in
enzymes that can bind and isomerize substrates
into higher affinity compounds.
• In previous work, palatinose, turanose and
fructose have been shown to inhibit MalA in a
time-dependent manner.
Palatinose
[Palatinose]
%inhib KI (mM)
initial final initial final
10 mM 48 76 6.6 1.9
20 mM 61 79 3.8 1.7
Fig. 7 Hydrolysis of pNPG by purified MalA in the presence and
absence of palatinose. The enzyme was inhibited in a time-
dependent manner consistent with previous observations.
Future Direction
Ironing Out Purification Protocol Issues
• Compared to previous purification attempts,
the anion-exchange step was ineffective.
Fig. 8 SDS-PAGE analysis of enzyme prep aliquots at various
stages of purification: filtered cell lysate (1:2 dilution; 15/20 µL,
lanes 1/2), after ion-exchange column (15/20 µL, lanes 3/4), MW
ladder (lanes 5/9), after hydrophobic interaction column (15/20
µL, lanes 6/7), BSA standard (lane 8).
HPLC: Quantifying Enzyme Activity
• Now that our HPLC system is operational, we
can use it to quantify hydrolysis products, and
definitively determine whether MalA has
glycosyltransferase or isomerase activities.