Exploring the effects of purine nucleotides on the structure and function of Burkholderia cenocepacia HMG-CoA reductase

Tara Cristallo, Matthew Clark, and Dr. Jeffrey WatsonDepartment of Chemistry and Biochemistry, Gonzaga University, 502 E. Boone Ave., Spokane WA 99258

Abstract

Introduction

Results

References and AcknowledgementsI. Lawrence, S.H. and Jaffe, E.K. (2008) Biochem. Mol. Biol. Educ. 36(4): 274-283II. Schwarz, B.H., Driver, J., Peacock, R.B., Dembinski, H.E., Corson, M.H., Gordon,II. S.S. and Watson, J.M. (2014) Biochem. Biophys. Acta 1844: 457-464lllllllllllllllll

This research was supported by a grant to Gonzaga University from the Howard Hughes Medical Institute through the Undergraduate Science Education Program, the Gonzaga Science Research Program, and the Anna Marie Ledgerwood Endowment.

HMG-Co A reductase (HMGR) is the key enzyme responsible for catalyzing the rate-determining step of the mevalonate pathway, one of two avenues for the biosynthesis of isoprenoids. The present study investigates the HMGR found in the bacterium Burkholderia cenocepacia (BcHMGR). Employment of kinetic assays have suggested that purine nucleotides can alter the kinetic activity of BcHMGR. Interestingly, it has been suggested that these molecules can result in enzymatic activity in the absence of one the necessary substrates for BcHMGR activity, namely Coenzyme A. 13C NMR and fluorescence spectroscopy are utilized to characterize possible differences between the two reaction products as well as probable structural changes in BcHMGR induced in the presence of purine nucleotides.

HMGR is a NAD(P)H-dependent enzyme that catalyzes the reversible four-electron reduction of HMG-CoA to mevalonate in the rate-determining step of the mevalonate pathway, one of two possible metabolic avenues for the biosynthesis of isoprenoids, an important class of biomolecules including cholesterol.

BcHMGR preferentially catalyzes the oxidation of mevalonate to HMG-CoA. B. cenocepacia, is an opportunistic lung pathogen capable of infecting compromised immune systems, causing often-fatal infections. BcHMGR has been shown to exhibit morpheein behavior resulting in unusual kinetic activity dictated by the presence of multiple different quaternary structures the enzyme can form. lllllllllllllllll

Previous research has suggested that purine nucleotide molecules can cause enzymatic activity of BcHMGR in the absence of the necessary substrate, Coenzyme A (CoA), resulting in NADH production (Figure 1A). NADH production is observed in the absence of CoA and presence of GTP and GDP but not in that of ADP. GTPase studies elucidate the production of free phosphate by BcHMGR, a result of the hydrolysis of purine nucleotides (Figure 1B). lllllllllllllllll

Employment of fluorescence spectroscopy allows for qualitative exploration of the structural changes resulting from the interaction between purine nucleotides and BcHMGR. 13C NMR permits investigation of changes in starting mevalonolactone in the presence of CoA or GTP to potentially elucidate a novel reaction for an HMGR.

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

Fluorescence spectroscopySamples were excited at 295 nm and monitored 320-400 nm using Cary Eclipse Fluorescence Spectrophotometer after each addition of 30 mM substrate stock to 0.5 or 1.0 mg/mL protein in desalt buffer pH 9. N-acetyl-L-tyrptophanamide (NATA) was subjected to identical analysis to serve as an analog for the study.lllllllllllllllll

13C NMR spectroscopy2.5 mg/mL BcHMGR in desalt buffer (HEPES, KCl) pH 7.5 was reacted with 60 mM NAD+, 90 mM mevalonolactone-5-13C, and 30 mM CoA. The reactants and protein were mixed in a 90% H2O/10% D2O desalt buffer. The reaction mixture was incubated for 5 minutes before being heated to 70°C for 20 minutes to denature the protein. The solution was then centrifuged through a 30,000 MWCO filter at 4,000 x g for 10 min to elute the reaction products. 13C NMR was performed with Bruker Topspin 2.0 and a H2O/D2O specific shim.

A B

A B

Methods

Discussion and Future WorkFluorescence spectroscopy studies• GTP induces a relatively unique structural change in BcHMGR • NAD+/H, ATP, and ADP appear to exert similar structural changes with respect to the

tryptophan residues• These findings could suggest GTP binds in a unique pocket of BcHMGR • Continue to investigate the affect of GDP and NAD+/H on the enzyme at pH 9

13C NMR studies• If present, mevalonolactone-5-13C stock is interconverting between the lactone

(67.8, 66.4 ppm) and mevalonate (58 ppm)

• However, absence of starting material carbonyl peak is not indicative of its presence• Dominating presence of buffer species, HEPES, (57.8, 49, 47 ppm, data not shown)

on product spectrum• Future work includes using an inorganic buffer

• Expected signal (180 ppm range) from the thioester-containing product, HMG-CoA, is not present in the product spectrum

• Upcoming steps will explore enzyme denaturation methods to release the product from the enzyme

• Usage of different deuterated solvents such as DMSO or CDCl3 could cause degradation of the enzyme and yield better visualization of the product.

Figure 2: Effect of purine nucleotides on BcHMGR. (A) Intrinsic tryptophan fluorescence of BcHMGR (0.5 mg/mL) 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) at pH 9. GTP results in near total quenching of fluorescence at 3.7 mM ligand. Figure 4: Conversion of

mevalonolactone (left) with mevalonate (right). The 13C-labeled carbon atom monitored in the assay is highlighted.

Figure 3: 13C NMR spectroscopy. (A) 90 mM mevalonolactone-5-13C in desalt buffer pH 7.5. Both lactone (67.8, 66.4 ppm) and mevalonate (58 ppm) are seen. (B) Filtered products from reaction of BcHMGR with mevalonolactone-5-13C, CoA, and NAD+. A lack of a peak in 180 ppm range indicates no formation of the product, HMG-CoA.

A

B

OO

OH

OHO

O OH

Na

Figure 1: Effect of purine nucleotides on the kinetic behavior of BcHMGR. (A) Kinetic assay monitoring NADH production in the presence of purine nucleotides. (B) Absorbance of free phosphate produced from GTPase activity of BcHMGR.