1. Exploration of the Structure and Function of Burkholderia
cenocepacia HMG-CoA Reductase
Tara Cristallo*, Alex Cumings*, Chad Hicks*, Matt Clark*, Riley Peacock*,
Alexander Walkerǂ, Dr. ChulHee Kangǂ, and Dr. Jeff Watson*
*Department of Chemistry and Biochemistry, Gonzaga University, 502 E. Boone Ave., Spokane WA 99258
and ǂDepartment of Chemistry, Washington State University, Pullman, WA 99164
Abstract
Introduction
Results
References and Acknowledgements
Lawrence, S.H. and Jaffe, E.K. (2008) Biochem. Mol. Biol. Educ. 36(4): 274-283
Schwarz, B.H., Driver, J., Peacock, R.B., Dembinski, H.E., Corson, M.H., Gordon, S.S. and
Watson, J.M. (2014) Biochem. Biophys. Acta 1844: 457-464
This research was supported in part by a grant to Gonzaga University from the Howard
Hughes Medical Institute through the Undergraduate Science Education Program. This
research was supported in part by the Gonzaga Science Research Program. This research
was supported in part by the Gonzaga Science Research Program and the Anna Marie
Ledgerwood Endowment.
HMG-CoA reductase (HMGR) in most organisms catalyzes the key
regulatory step in the reductive biosynthesis of isoprenoids via the
mevalonate pathway. However, the opportunistic lung pathogen
Burkholderia cenocepacia lacks the other enzymes of the
mevalonate pathway and uses its HMGR oxidatively as part of an
unknown metabolic pathway. B. cenocepacia HMGR (BcHMGR)
also exhibits many properties characteristic of a morpheein, in
which the enzymatic activity is regulated by a complex equilibrium
between quaternary structures that each possess different
intrinsic levels of enzyme activity. The equilibrium between states
is controlled by changes in ligand concentration, enzyme
concentration, and solution pH.
HMGR in most organisms catalyzes the reversible four-electron
reduction of the thioester HMG-CoA to the primary alcohol
mevalonate, using NAD(P)H as the hydride carrier.
BcHMGR and at least one other bacterial HMGR preferentially
catalyze the oxidation of mevalonate to HMG-CoA. However,
BcHMGR exhibits kinetic and structural behavior characteristic of a
morpheein protein. In the morpheein model of allostery
(Lawrence and Jaffe, 2008), a homooligomeric protein dissociates
into a lower order state, undergoes a tertiary structure change,
and reassembles into a different, nonadditive quaternary form
(Figure 1a). These quaternary forms exhibit different levels of
enzymatic activity, leading to unusual kinetic behavior (Figure 1b,
Schwarz et al. 2014) due to the presence of multiple states of the
enzyme in solution. The equilibrium between the multiple
oligomeric states can be governed by many factors, including
ligand/substrate concentration, enzyme concentration and
solution pH. In addition, morpheeins often exhibit more than one
enzymatic activity.
O
O OHCH3
O
SCoA O
O CH3 OH OH
SCoA O
O CH3 OH O
H O
O CH3 OH OH
H
(R)-mevalonate
NAD(P)+
(S)-HMG-CoA [mevaldyl-CoA] [mevaldehyde]
NAD(P)H
NAD(P)+
NAD(P)H
CoASH
Figure 1: The morpheein model of allostery. (A) Schematic of a
morpheein protein. Multiple oligomeric states are present at once, in
equilibrium with one another. (B) The presence of multiple active states
in solution leads to multiple saturation kinetic behavior (shown for
BcHMGR)
A B
Figure 2: Size exclusion chromatography of BcHMGR. (A) Effect of pH on oligomeric
distribution. Peaks correspond to 18-mer, nonamer and hexamer from left to right,
respectively. All runs utilized 6 mg/mL BcHMGR. (B) 100 μL of 4.014 mg/mL BcHMGR was
qualitatively analyzed at pH 7.5. Three unique oligomeric states are evident on the
spectrum (three green peaks) of which further characterization must be done to
quantitatively identify each state.
Figure 3: Effect of purine nucleotides on BcHMGR. (A) Intrinsic tryptophan fluorescence
of BcHMGR (0.5 mg/mL) vs. ligand concentration at pH 7.5. GTP and GDP result in near
total quenching of fluorescence at 5 mM ligand. (B) Intrinsic tryptophan fluorescence of
BcHMGR (0.5 and 1.0 mg/mL) vs. ligand concentration at pH 9. Similar to results obtained
at pH 7.5, GTP condition results in near total quenching of fluorescence as low as 3.7 mM
ligand.
A B
C
Figure 4: Crystal structures of BcHMGR, pH 7.5 + 10 mM CoA. (A) Asymmetric unit. Three
monomers of BcHMGR form the asymmetric unit of a dimer plus one monomer. (B)
Asymmetric unit. Coenzyme A is bound to one monomer, while ADP is bound to a second
monomer. The third remains empty in this structure. (C) Crystallographic hexamer.
Symmetry-related chains are shown in bright blue.
pH
Ligands
Added
Ligands
Present
Resolution
7.5 10 mM CoA
CoA (1),
ADP? (1)
2.05 Å
8.3 10 mM GTP
CoA (3),
NDP (2)
2.8 Å
9.0
10 mM CoA,
10 mM GTP
CoA (3),
GTP (1),
NDP (2)
2.3 Å
A B
Figure 4: GTPase activity of BcHMGR measured by luciferase luminescence. The
presence of GTP leads to luminescence. Luminescence of the enzyme-present reaction
is compared to the luminescence of a positive control enzyme-absent reaction to give
a relative luminescence. Higher GTPase activity is depicted by lower rel.
luminescence. (A) GTPase activity of 1.00 mg/mL BcHMGR in the presence and
absence of mevalonate and NAD+. (B) GTPase activity of 0.25 mg/mL BcHMGR in the
presence and absence of mevalonate and NAD+.
Future plans include calibrating the SEC-MALS instrument to minimize error in order to
characterize oligomers of BcHMGR. Samples of purified protein under different conditions
varying pH, protein concentration, presence of substrates (NAD+, coenzyme A (CoA),
mevalonate) and ligands (GTP, GDP, ATP and ADP) will also be investigated. Fluorescence
experiments will be conducted to explore the effects of NAD+, NADH, and GDP on BcHMGR at
pH 9. Further GTPase activity of BcHMGR will be studied in the presence of different
combinations of mevalonate, NAD+, and CoA. Enzyme hydrolysis and free phosphate
production of GDP, ADP, and ATP will also be explored.
Future Work
A B
A B