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 99258and ǂDepartment of Chemistry, Washington State University, Pullman, WA 99164

Abstract

Introduction

Results

References and AcknowledgementsLawrence, S.H. and Jaffe, E.K. (2008) Biochem. Mol. Biol. Educ. 36(4): 274-283Schwarz, 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