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Protein Science (2004), 13:1693-1697. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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FOR THE RECORD

Inhibition of the Class II HMG–CoA reductase of Pseudomonas mevalonii

Matija Hedl and Victor W. Rodwell

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-2063, USA

Reprint requests to: Victor W. Rodwell, Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, IN 47907-2063, USA; e-mail: vrodwell{at}purdue.edu; fax: (765) 494-7897.

(RECEIVED December 22, 2003; FINAL REVISION March 9, 2004; ACCEPTED March 14, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
There are two structural classes of HMG–CoA reductase, the third enzyme of the mevalonate pathway of isopentenyl diphosphate biosynthesis—the Class I enzymes of eukaryotes and the Class II enzymes of certain eubacteria. Structural requirements for ligand binding to the Class II HMG–CoA reductase of Pseudomonas mevalonii were investigated. For conversion of mevalonate to HMG–CoA the –CH3, –OH, and –CH2COO– groups on carbon 3 of mevalonate were essential for ligand recognition. The statin drug Lovastatin inhibited both the conversion of HMG–CoA to mevalonate and the reverse of this reaction. Inhibition was competitive with respect to HMG–CoA or mevalonate and noncompetitive with respect to NADH or NAD+. Ki values were millimolar. The over 104-fold difference in statin Ki values that distinguishes the two classes of HMG–CoA reductase may result from differences in the specific contacts between the statin and residues present in the Class I enzymes but lacking in a Class II HMG–CoA reductase.

Keywords: HMG-CoA reductase; Class II HMG-CoA reductase; Pseudomonas mevalonii; isoprenoid biosynthesis; statin drug; Lovastatin

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03597504.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
HMG–CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase) is the rate-limiting enzyme of the mevalonate pathway of isopentenyl diphosphate synthesis. Catalysis by this 4-electron oxidoreductase proceeds in three stages, the first and third of which are reductive. HMG–CoA reductases from eukaryotes, archaea, and most bacteria use NADPH, whereas the enzyme from Pseudomonas mevalonii and certain other bacteria employ NADH (equation 1):


(1)

The reaction is freely reversible (equation 2):


(2)

Inspection of sequence alignments of HMG–CoA reductases from eukaryotes, archaea, and bacteria revealed two classes of HMG–CoA reductase (Bochar et al. 1999). Eukaryotes (Frimpong and Rodwell 1994) and most archaea (Bochar et al. 1997) elaborate Class I HMG–CoA reductases, whereas the HMG–CoA reductases of Pseudomonas mevalonii (Jordan-Starck and Rodwell 1989), Staphylococcus aureus (Wilding et al. 2000b), Enterococcus faecalis (Hedl et al. 2002), some streptomycetes (Takahashi et al. 1999; Dairi et al. 2000), and the archaeon Archaeoglobus fulgidus (Kim et al. 2000) belong to Class II. Crystal structures of statin drugs bound to HMG–CoA reductase are available for the Class I human enzyme (Istvan and Deisenhofer 2001) and for the Class II P. mevalonii enzyme (Tabernero et al. 2003). Whereas the inhibition of mammalian HMG–CoA reductase by statins has been extensively investigated (Alberts et al. 1980; Bischoff and Rodwell 1986; Endo 1992), inhibition by statins of the Class II P. mevalonii enzyme has not been reported. We here investigate inhibition of the Class II HMG–CoA reductase from P. mevalonii by mevalonate analogs and by Lovastatin.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Inhibition by substrate analogs
The inhibition of reaction 2, the oxidative acylation of mevalonate to HMG–CoA, by mevalonate analogs was investigated with respect to mevalonate. The compounds tested included {beta}-hydroxybutyrate, acetoacetate, 3-hydroxy-3-methylglutarate, {gamma}-hydroxybutyrate, 3-hydroxybu-tyrate methyl ester, 3-aminobutyrate, citrate, 1,3-butanediol, oxaloacetate, and threonine (Fig. 1Go). Double-reciprocal plots of these inhibition studies are shown in Figure 2Go. Table 1Go summarizes the inhibition constants and inhibition types.



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  		Figure 1. Structures of compounds tested for inhibition of P. mevalonii HMG–CoA reductase and of the substrates, HMG–CoA and mevalonate. (Top left) Positions of the indicated functional groups. (Top right) Structure of Lovastatin.

 


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  		Figure 2. Inhibition of P. mevalonii HMG–CoA reductase by substrate analogs. Assays of reaction 2, the oxidative acylation of mevalonate, were conducted under standard conditions except for the indicated concentrations of compounds tested as inhibitors. All reactions were initiated by adding mevalonate. Activities in the presence of an inhibitor are expressed relative to the maximal observed activity in the absence of inhibitor, the value at 0.50 mM mevalonate or at a 1/[S] of 2.0. (Top left) {beta}-hydroxybutyrate. Assays employed 0 mM (filled circles), 0.65 mM (open circles), 1.25 mM (filled squares), or 2.5 mM (open squares) {beta}-hydroxybutyrate. (Top right) Acetoacetate. Assays employed 0 mM (filled circles), 0.65 mM (open circles), 1.25 mM (filled squares), or 2.5 mM (open squares) acetoacetate. (Bottom left) 3-Hydroxy-3-methylglutarate (HMG). Assays employed 0 mM (filled circles), 12.5 mM (open circles), 25 mM (filled squares), or 50 mM (open squares) 3-hydroxy-3-methylglutarate. (Bottom right) {gamma}-Hydroxybutyrate. Assays employed 0 mM (filled circles), 25 mM (open circles), 50 mM (filled squares), or 100 mM (open squares) {gamma}-hydroxybutyrate.

 

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  		Table 1. Inhibitors of P. mevalonii HMG–CoA reductase
 
Inhibition by Lovastatin
Inhibition by Lovastatin of reaction 2, the oxidative acylation of mevalonate to HMG–CoA, and of reaction 1, the reductive deacylation of HMG–CoA to mevalonate, was investigated with respect to mevalonate, NADH, NAD+, and HMG–CoA. Double-reciprocal plots are shown in Figure 3Go. Table 2Go summarizes inhibition constants and types of inhibition.



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  		Figure 3. Inhibition by Lovastatin of P. mevalonii HMG–CoA reductase. (Top) Inhibition of reaction 2, the oxidative acylation of mevalonate to HMG–CoA. Reactions were initiated by adding mevalonate. (Bottom) Inhibition of reaction 1, the reductive deacylation of HMG–CoA to mevalonate. Reactions were initiated by adding HMG–CoA. Activities in the presence of Lovastatin are expressed relative to the maximal observed activity in the absence of Lovastatin, the value at 0.50 mM substrate or coenzyme or at a 1/[S] or 1/[CoE] of 2.0. For both reactions, assays employed 0 (filled circles), 6.5 mM (open circles), 13 mM (filled squares), and 26 mM (open squares) Lovastatin under otherwise standard conditions.

 

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  		Table 2. Inhibition of P. mevalonii HMG–CoA reductase by Lovastatin
 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Prior investigations of substrate analog inhibition of the HMG–CoA reductases of two soil isolates (Fimognari and Rodwell 1965) and of the human enzyme (Karl et al. 1995) implicated the –CH3 and –OH groups on the {beta}-carbon and the carboxyl group of mevalonate in ligand recognition. An ethyl group can, however, replace the 3-methyl group for the HMG–CoA reductase of rat liver and of the moth Manduca sexta (Baker and Schooley 1981). {beta}-Hydroxybutyrate, acetoacetate, 3-hydroxy-3-methylglutarate, and {gamma}-hydroxy-butyrate inhibited P. mevalonii HMG–CoA reductase competitively with respect to mevalonate. However, none had a Ki lower than the Km value of 0.26 mM for mevalonate (Rodwell et al. 2000). 3-Hydroxybutyrate methyl ester, 3-aminobutyrate, citrate, 1,3-butanediol, oxaloacetate, and threonine did not significantly inhibit activity. Structurally, all the inhibiting compounds except {gamma}-hydroxybutyrate contained the –CH3, –OH, or carbonyl, and –CH2COO– groups on the {beta}-carbon. Substitutions of any of these groups resulted in loss of significant inhibition.

Lovastatin, a member of the "statin" group of drugs that are potent inhibitors of human HMG–CoA reductase, shares structural features with HMG–CoA. Lovastatin contains the carboxyl group and the –OH group on the {beta}-carbon but lacks the methyl group on the {beta}-carbon of HMG–CoA. A hydrophobic moiety replaces the coenzyme A portion of HMG–CoA (Fig. 1Go). For reactions 2 and 1, inhibition of P. mevalonii HMG–CoA reductase by Lovastatin was competitive with respect to mevalonate or HMG–CoA and non-competitive with respect to NAD+ or NADH. This statin drug therefore mimics binding of the substrates but not of the coenzymes. For inhibition of reaction 1 with respect to HMG–CoA, the 0.53 mM Ki for Lovastatin was approximately 104-fold higher than the nanomolar values for the Class I enzymes (Alberts et al. 1980; Bischoff and Rodwell 1996). Comparison of the crystal structures of statins bound to the human enzyme (Istvan and Deisenhofer 2001) and to the enzyme from P. mevalonii (Tabernero et al. 2003) suggested that their characteristic Ki values may be due to the differences in the specific contacts between the statins and particular residues of the Class I and Class II enzymes (Tabernero et al. 2003). As the survival of many Gram-positive pathogens requires a functional Class II HMG–CoA reductase (Wilding et al. 2000a, b), it thus ultimately may be feasible to exploit differences between the structures of the two classes of this enzyme to design inhibitory antibiotics directed against the Class II enzymes of multi-drug resistant bacteria.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Reagents
Mevalonolactone, (R,S)-HMG–CoA, {beta}-NADH, {beta}-NAD+, (R,S)-{beta}-hydroxybutyrate, acetoacetate, (R,S)-3-hydroxy-3-methylglutarate, {gamma}-hydroxybutyrate, (R)-3-hydroxybutyrate methyl ester, (R,S)-3-aminobutyrate, citrate, (R,S)-1,3-butanediol, oxaloacetate, (R,S)-threonine, and Lovastatin were purchased from Sigma.

Protein expression and purification
The hmgr gene of pHMGR (pKK177-3-RED) that encodes P. mevalonii HMG–CoA reductase (Beach and Rodwell 1989) was expressed in Escherichia coli BL21(DE3) cells (Novagen). HMG–CoA reductase was then purified to homogeneity as described previously (Rodwell et al. 2000).

HMG–CoA reductase activity
Spectrophotometric assays of P. mevalonii HMG–CoA reductase activity at 37°C monitored the appearance or disappearance of NADH at 340 nm. Standard assay conditions were as follows: (1) Reaction 1, reductive deacylation of HMG–CoA to mevalonate: 0.5 mM NADH, 1.0 mM (R,S)-HMG–CoA, 100 mM KCl, and 100 mM KiPO4 (pH 6.5); (2) Reaction 2, oxidative acylation of mevalonate to HMG–CoA: 4 mM NAD+, 2 mM coenzyme A, 6 mM (R,S)-mevalonate, 100 mM KCl, and 100 mM Tris-HCl (pH 8.0). Assays employed a final volume of 200 µL and were initiated by the addition of mevalonate or HMG–CoA. The rate of reaction was then measured for 10 s. One enzyme unit (eu) represents the turnover, in 1 min, of 1 µmole of NAD(H). This corresponds to the turnover of 0.5 µmole of HMG–CoA or mevalonate. Reported results represent mean values for at least duplicate determinations. Inhibition constants for competitive inhibition were derived from the slopes of Lineweaver-Burke plots using the formula 1/v = (Km/Vmax)(1/[S])(1 + [I]/Ki) + 1/Vmax. Inhibition constants for the noncompetitive inhibition were derived from the y-intercepts of Lineweaver-Burke plots using the formula 1/Vmax = 1 + [I]/Ki/v. Ki values were calculated for each concentration of inhibitor in a single experiment and averaged.


   Acknowledgments
 
Journal paper 17098 from the Purdue University Agricultural Experiment Station. This research was funded by American Heart Association grant 0150503N. The data are from the Ph.D. thesis of Matija Hedl. We thank Lydia Tabernero for information and advice regarding crystal structures of Lovastatin bound to P. mevalonii HMG–CoA reductase.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Alberts, A.W., Chen, J., Kuron, G., Hunt, V., Huff, J., Hoffman, C., Rothrock, J., Lopez, M., Joshua, H., Harris, E., et al. 1980. Mevinolin: A highly potent competitive inhibitor of hydroxymethylglutaryl–coenzyme A reductase and a cholesterol-lowering agent. Proc. Nat. Acad. Sci. 77: 3957–3961.[Abstract/Free Full Text]

Baker, F.C. and Schooley, D.A. 1981. Biosynthesis of 3-hydroxy-3-methylglutaryl-CoA, 3-hydroxy-3-ethylglutaryl-CoA, mevalonate and homomevalonate by insect corpus allatum and mammalian hepatic tissues. Biochim. Biophys. Acta 664: 356–372.[Medline]

Beach, M.J. and Rodwell, V.W. 1989. Cloning, sequencing, and overexpression of mvaA, which encodes Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase. J. Bacteriol. 171: 2994–3001.[Abstract/Free Full Text]

Bischoff, K.M. and Rodwell, V.W. 1996. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: Purification, characterization, and expression in Escherichia coli. J. Bacteriol. 178: 19–23.[Abstract/Free Full Text]

Bochar, D.A., Brown, J.R., Doolittle, W.F., Klenk, H.P., Lam, W., Schenk, M.E., Stauffacher, C.V., and Rodwell, V.W. 1997. 3-Hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product. J. Bacteriol. 179: 3632–3638.[Abstract/Free Full Text]

Bochar, D.A., Stauffacher, C.V., and Rodwell, V.W. 1999. Sequence comparisons reveal two classes of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mol. Genet. Metab. 66: 122–127.[CrossRef][Medline]

Dairi, T., Motohira, Y., Kuzuyama, T., Takahashi, S., Itoh, N., and Seto, H. 2000. Cloning of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from terpenoid antibiotics-producing Streptomyces strains. Mol. Gen. Genet. 262: 957–964.[CrossRef][Medline]

Endo, A. 1992. The discovery and development of HMG–CoA reductase inhibitors. J. Lipid. Res. 33: 1569–1582.[Medline]

Fimognari, G.M., and Rodwell, V.W. 1965. Substrate-competitive inhibition of bacterial mevalonate:nicotinamide adenine dinucleotide oxidoreductase (acylating CoA). Biochemistry 4: 2086–2090.[CrossRef]

Frimpong, K. and Rodwell, V.W. 1994. Catalysis by Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proposed roles of histidine 865, glutamate 558, and aspartate 766. J. Biol. Chem. 269: 11478–11483.[Abstract/Free Full Text]

Hedl, M., Sutherlin, A.L., Wilding, E.I., Mazzulla, M., McDevitt, D., Lane, P., Burgner 2nd, J.W., Lehnbeuter, K.R., Stauffacher, C.V., Gwynn, M.N., et al. 2002. Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis. J. Bacteriol. 184: 2116–2122.[Abstract/Free Full Text]

Istvan, E.S. and Deisenhofer, J. 2001. Structural mechanism for statin inhibition of HMG–CoA reductase. Science 292: 1160–1164.[Abstract/Free Full Text]

Jordan-Starck, T.C. and Rodwell, V.W. 1989. Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase characterization and chemical modification. J. Biol. Chem. 264: 17913–17918.[Abstract/Free Full Text]

Karl, R.M., Hupperich, M., Thomer, A., and Eggerer, H. 1995. Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase and citrate synthase by sulfoxides and sulfones of substrate-analogue CoA-thioether derivatives. Eur. J. Biochem. 227: 292–295.[Medline]

Kim, D.-Y., Stauffacher, C.V., and Rodwell, V.W. 2000. Dual coenzyme specificity of Archaeoglobus fulgidus HMG–CoA reductase. Protein Sci. 9: 1226–1234.[Abstract]

Rodwell, V.W., Beach, M.J., Bischoff, K.M., Bochar, D.A., Darnay, B.G., Friesen, J.A., Gill, J.F., Hedl, M., Jordan-Starck, T.C., Kennelly, P.J., et al. 2000. 3-Hydroxy-3-methylglutaryl-CoA reductase. Methods Enzymol. 324: 259–280.[CrossRef][Medline]

Tabernero, L., Rodwell, V.W., and Stauffacher, C.V. 2003. Crystal structure of a statin bound to a class II HMG–CoA reductase. J. Biol. Chem. 278: 19933–19938.[Abstract/Free Full Text]

Takahashi, S., Kuzuyama, T., and Seto, H. 1999. Purification, characterization, and cloning of a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in biosynthesis of terpenoids. J. Bacteriol. 181: 1256–1263.[Abstract/Free Full Text]

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