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Thermostable archaeal HMG -CoA reductases: Regulation of the activity of Sulfolobus solfataricus HMG-CoA reductase by reversible phosphorylation and dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase
No full textThe activity of the HMG-CoA reductases of higher eukaryotes is regulated by reversible phosphorylation of a putative active site serine. Mutagenic and kinetic evidence suggested that interaction between phosphoserine and the catalytic histidine, negates the ability of the histidine to protonate the departing coenzyme A thioanion. However, physical evidence for this interaction is lacking. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus contains alanine (Ala406) in place of a phosphorylatable serine. Its activity thus is not regulated by phosphorylation. In addition to providing long-term stability (retains activity above 80°C), a feature desirable both for physical studies and for possible exploitation as an industrial biocatalyst, this enzyme therefore represents a good candidate for crystallographic investigation. A form of S. solfataricus HMG-CoA reductase whose activity was regulated by reversible phosphorylation was engineered by replacing Ala406 by serine and introducing a cAMP-dependent protein kinase recognition motif. Ultimate solution of the three-dimensional structures of its phospho- and dephosphoforms could therefore reveal structural features critical for the phosphorylation-mediated regulation of HMGCoA reductase activity. The inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus suggested that it might encode a Class II HMG-CoA reductase. Following PCR-based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85°C. Mevinolin inhibited competitively with HMG-CoA. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four catalyzed reactions revealed that optimal activity using NADP(H) occurred at a pH 1 to 3 units more acidic than that using NAD(H). Kinetic parameters were determined for all four reactions using either NAD(H) or NADP(H). These coenzymes compete for a common site. kcat[NAD(H)]/k cat[NADP(H)] varied from 1 to under 70 for the four reactions, indicative of a slight preference for NAD(H). Km and kcat values as a function of pH suggested that the protonated forms of two residues, probably His390 and Lys277, are essential for activity
The mevalonate pathway of isopentenyl pyrophosphate biosynthesis in Enterococcus faecalis: A potential target for antimicrobial agents
No full textBiosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multi-drug resistant strains of Gram-positive cocci employ the mevalonate pathway for IPP biosynthesis (E. I. Wilding et al. Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in Gram-positive cocci. J. Bacteriol. 182:4319, 2000). Bacterial mevalonate pathway enzymes therefore offer potential targets for development of inhibitors for use as antibiotics. Enterococci possess an open reading frame, mvaE, that appears to encode two catalytic centers of the mevalonate pathway, acetoacetyl coenzyme A thiolase and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium and Enterococcus hirae. mvaE was cloned from Enterococcus faecalis and expressed with an N-terminal histidine tag in Escherichia coli. The 86.5 kDa mvaE gene product, purified by nickel affinity chromatography, catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, ΔH a, Km values and pH optima were determined for acetoacetyl-CoA thiolase. Kinetic studies implicated a ping-pong mechanism. Coenzyme A inhibited competitively with acetyl-CoA. The polymerase chain reaction and E. faecalis genomic DNA were used to isolate the mvaS gene that encodes HMG-CoA synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in E. coli with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni2+-agarose to apparent homogeneity (specific activity 10 μmoles/min/mg). The enzyme is a dimer, mass 83.9 kDa, S20w 5.3. Optimal activity occurred at pH 9.8 in 2.0 mM MgCl2 at 37°C. ΔHa was 6000 calories per mole. The stoichiometry per monomer of acetyl-CoA binding was 1.2 ± 0.2 and of covalent acetylation was 0.60 ± 0.02. Km for the hydrolysis of acetyl-CoA was 10 μM. Coupled conversion of acetyl-CoA to mevalonate was achieved using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis. A hollow fiber reactor and NiNTA affinity support were used to trap the fusion protein, HMG-CoA synthase, and unfused HMG-CoA reductase from E. faecalis to form a bioreactor that converted acetyl-CoA to mevalonate
Purification, characterization, and gene cloning of one subunit of the heteromeric transcriptional activator, MvaT, of the Pseudomonas mevaloniimvaAB operon
No full textRegulation of the Pseudomonas mevalonii mvaAB operon that encodes HMG-CoA reductase (E.C. 1.1.1.88) and HMG-CoA lyase (E.C. 4.1.3.4) occurs at the level of transcription. The gene products of the mvaAB operon facilitate the first two steps in the catabolism of mevalonate. MvaT, the transcriptional activator of the mvaAB operon, has been purified over 1,200-fold and partially characterized. MvaT is expressed constitutively and binds in vitro to the regulatory cis-element upstream of the mvaAB operon with a K\rm\sb{d} \sb{app} of 2 nM. Mevalonate is necessary for activation of transcription in vivo, however, in vitro binding of MvaT to the cis-element occurs in the absence of mevalonate. Purification of MvaT enriched for 2 dissimilar polypeptides of approximate molecular mass 15 and 16 kDa. These polypeptides were termed P15 and P16, respectively. P15, P16, and MvaT activity comigrated during extensive purification that included DNA-affinity and size exclusion chromatography, and sucrose density gradient centrifugation. P15 and P16 also comigrated when purified MvaT was subjected to denaturing isoelectric focusing. Treatment of purified MvaT with the crosslinking reagent dimethylsuberimidate resulted in the formation of a 31 kDa polypeptide complex that contained amino acid sequence from P15 and P16. The apparent association of P15 and P26 in solution and their comigration with MvaT suggests that MvaT is a heteromeric protein composed of P15 and P16 subunits. The molecular mass of MvaT, estimated by size exclusion chromatography, was approximately 33 kDa, a value consistent with MvaT being a dimeric protein composed of P15 and P16. A portion of the gene that encodes P16 was cloned from P. mevlonii using a polymerase chain reaction approach that utilized degenerated PCR primers directed against the N-termini of P15 and P16. The deduced amino acid sequence of P16 predicts a polypeptide having a molecular weight of 14,300 and estimated by computer software to have a pI of approximately 10. As judged by codon frequency, codon usage in the P16 gene is essentially identical to that of mvaA and mvaB
General acid/base catalysis by a histidine residue of mammalian and bacterial HMG-CoA reductase
No full textBy investigating the pH-variation of kinetic parameters, Veloso, Cleland, and Porter (Biochemistry 20, 887-894, 1981) postulated that a histidine residue participates in catalysis by yeast 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Histidine modifying reagents also inactivate yeast HMG-CoA reductase (Dugan and Katiyar, Biochem. Biophys. Res. Commun. 141, 278-284, 1986). I therefore used chemical modification and site-directed mutagenesis to identify a catalytic site histidine of HMG-CoA reductase of Pseudomonas mevalonii (Ps. HMGR) and of the catalytic domain of the Syrian hamster enzyme (R\sb{\rm cat}). Ps. HMGR and R\sb{\rm cat} were overexpressed in Escherichia coli, purified to homogeneity, and characterized. Diethyl pyrocarbonate (DEPC) inactivated both enzymes, and hydroxylamine partially restored activity. Sequence comparisons revealed that only His\sp{381} (Ps. HMGR) and His\sp{865} (R\sb{\rm cat}) are totally conserved among the catalytic domains of all known HMG-CoA reductases. The codon for His\sp{381} was changed to the codons for alanine, lysine, asparagine, and glutamine, and that for His\sp{865} to the codons for lysine and glutamine. Following overexpression in E. coli, all mutant enzymes were purified to homogeneity. While all mutant enzymes exhibited less than 1.5% of wild-type catalytic activity, all chromatographed on substrate affinity supports like wild-type enzyme, and K\sb{\rm m} values approximated those for wild-type enzyme. In addition, Ps. HMGR mutant enzymes exhibited wild-type crystal morphology. I therefore infer that the low catalytic activity of these mutant enzyme was not a result of gross conformational changes. Whereas His\sp{381} mutant enzymes were not inactivated by DEPC, His\sp{865} mutant enzymes were sensitive to DEPC. pK\sb1 values were determined for all Ps. HMGR mutant enzymes. Mutant enzyme Ps. HMGR H381K exhibited an elevated pK\sb1 of 10.2, consistent with lysine acting as a general base at high pH. Exogenous amines enhanced the activity of Ps. HMGR H381A in a pH-dependent manner, suggesting that the unprotonated amine acts as the general base in catalysis. His\sp{381} of Ps. HMGR and His\sp{865} of R\sb{\rm cat}, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xxx-Xxx-Asn-Arg-Ser motif of the catalytic domain of all eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis
HMG-CoA reductase: The active site lysine, the Sulfolobus solfataricus enzyme, and cloning of genes for two class II enzymes
No full textIn eukaryotes and archaea 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes an early, rate-limiting reaction in isoprenoid biosynthesis that in humans is the target of drugs that reduce serum cholesterol levels. The only available crystal structure is that of the biodegradative enzyme from Pseudomonas mevalonii. This structure allowed the identification of an active site lysine, lysine 267. Mutation of lysine 267 to alanine, arginine, aspartate, histidine, or cysteine was accompanied by loss of detectable activity. Introduction of the non-naturally occurring amino acid aminoethylcysteine resulted in full recovery of activity. The future characterization of this modified enzyme will be important in the investigation of the role of lysine 267 in catalysis. In an attempt to identify in the enzyme from Syrian hamster the cognate residue of P. mevalonii lysine 267, lysines 690, 691, and 734 were mutated to alanine. While sequence alignments suggest lysine 734 as the active site lysine, the mutagenesis results did not allow the unequivocal assignment of the cognate residue. While the three mutant enzyme had detectable activity, all appeared impaired for catalysis with specific activities for enzymes K690A, K691A, and K734A of 0.2%, 1.6%, and 0.16% wild-type activity. Solution of a crystal structure of a biosynthetic HMG-CoA reductase would allow the identification of the active site lysine. The gene ( hmgA) for HMG-CoA reductase of the archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus hmgA encodes a biosynthetic enzyme that was purified in 40% yield (specific activity 17.5 μU/mg at 50°C). The substrates are (S)-HMG-CoA (Km 17 μM) and NADPH (Km 23 μM). The enzyme was stable at 90°C and optimally active at pH 5.5 and 85°C. The thermostability of this enzyme makes it a good candidate for crystallization. Comparison of 50 sequences revealed two classes of HMG-CoA reductase. The catalytic domain of the human enzyme and the enzyme from P. mevalonii are proposed as canonical sequences for Class I and Class II HMG-CoA reductases, respectively. Only one Class II enzyme has previously been cloned, the biodegradative HMG-CoA reductase from P. mevalonii. Two new, apparently biosynthetic, Class II HMG-CoA reductase genes were cloned, from the eubacterium and Borrelia burgdorferi from the archaeon Archaeoglobus fulgidus
Protein engineering of Pseudomonas mevalonii HMG-CoA reductase determinants of nucleotide coenzyme specificity and conversion to a form whose activity is regulated by phosphorylation/dephosphorylation
No full text3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (E.C. 1.1.1.88) from the soil bacterium Pseudomonas mevalonii, an enzyme not naturally regulated by phosphorylation, utilizes NAD\sp+ to oxidize mevalonate to HMG-CoA. By contrast, mammalian HMG-CoA reductases are regulated by phosphorylation and utilize NADPH to reduce HMG-CoA to mevalonate. P. mevalonii HMG-CoA reductase was engineered to confer the following properties: (1) Improved use of the unnatural coenzyme NADP\sp+ and (2) Regulation by reversible phosphorylation-dephosphorylation. The 550,000-fold preference of P. mevalonii HMG-CoA reductase for NAD\sp+ versus NADP\sp+ was reduced to just a 7-fold preference by simultaneously substituting alanine for aspartate 146, which appears to hydrogen-bond to the 2\sp\prime-hydroxyl of the adenine ribose of NAD\sp+, and changing adjacent residue leucine148 to arginine. The activity of HMG-CoA reductases of higher eukarya is regulated by reversible phosphorylation of a putative active site serine. Since in P. mevalonii HMG-CoA reductase arginine387 replaces the phosphoacceptor serine, activity is not regulated by phosphorylation. An initial attempt to engineer forms of P. mevalonii HMG-CoA reductase whose activity is modulated by phosphorylation by constructing P. mevalonii-hamster chimeric enzymes yielded chimera that were phosphorylatable but inactive. Arginine387 was then replaced by serine, generating an active enzyme, but one that was not readily phosphorylated. Multiple amino acid residues around serine387 were next mutated, revealing combinations of amino acids critical for phosphorylation of HMG-CoA reductase by HMG-CoA reductase kinase. This generated two active mutant enzymes into which phosphate could be incorporated whose activity was reversibly modulated by phosphorylation-dephosphorylation. This appears to represent the only known instance where an enzyme has been engineered to a form whose activity can be modulated by phosphorylation-dephosphorylation. That the reversible phosphorylation models mammalian resembles that of the mammalian enzyme. The structure of a P. mevalonii HMG-CoA reductase whose activity is modulated by incorporation and release of phosphate should ultimately provide a structural view of the mechanism whereby the activity of HMG-CoA reductase is attenuated by phosphorylation
Isopentenyl pyrophosphate synthesis in bacteria: Genes and enzymes of the mevalonate pathway
No full textMany bacteria employ the non-mevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, Gram-positive cocci and Borrelia burgdorferi use exclusively the mevalonate pathway, which is essential for their growth (Wilding, E. I., Kim, D-Y., Bryant, A. P., Gwynn, M. N., Lunsford, R. D., McDevitt, D., Myers, J. E., Jr., Rosenberg, M., Sylvester, D., Stauffacher, C. V., and Rodwell, V. W. 2000. Essentiality, expression and characterization of the Class II HMG-CoA reductase of Staphylococcus aureus. J. Bacteriol. 182:5147–5152). Enzymes of the mevalonate pathway thus are potential targets for drug intervention. The enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl coenzyme A thiolase and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium and Enterococcus hirae. The mvaE gene was cloned from Enterococcus faecalis and expressed with an N-terminal histidine tag in Escherichia coli. The gene product was purified by nickel affinity chromatography and catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Optimal pH and temperature, ΔHa, and Km values were determined for HMG-CoA reductase activity. A millimolar Ki for a statin drug confirmed that E. faecalis HMG-CoA reductase is a Class II enzyme. The oxidoreductant was NADP(H). Consistent with participation of a histidine during stage one of the HMG-CoA reductase reaction, diethylpyrocarbonate blocked formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases implicated this histidine as His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme. The mvaK1 gene encoding E. faecalis mevalonate kinase was PCR-cloned and expressed with a C-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. Temperature and pH optima, ΔHa, and Km values were determined. Mevalonate kinase exhibits broad phosphoryl donor specificity. The K i for inhibition by ADP with respect to ATP was 2.7 mM. The characterization of the two bacterial enzymes of the mevalonate pathway is potentially important in the development of antibiotics against pathogens
Mevalonate catabolism in Pseudomonas: Cloning and sequencing of HMG -coenzyme A reductase
No full textProtein sequencing of HMG-CoA reductase from Pseudomonas sp. M gave 45% of the predicted structure from which two specific oligonucleotide probes were designed for use in cloning the gene. The probes were used to screen a restriction fragment-enriched Pseudomonas:pUC19 library. The positive clone obtained was then used as a probe for screening a Pseudomonas:EMBL4 library from which a 14 kb positive insert was obtained. A 2.1 kb PstI/HindIII fragment was sequenced and shown to encode HMG-CoA reductase based on comparison to the cyanogen bromide peptide amino acid composition and sequence data. The HMG-CoA reductase gene encodes a hydrophilic protein of MW 45,538. Comparison of Pseudomonas sp. M and mammalian HMG-CoA reductases gives two blocks of homology with approximately 33% homology over 29% of the Pseudomonas enzyme sequence, which may reflect their functional relatedness. Comparison of cysteine-containing peptides in Pseudomonas sp. M HMG-CoA reductase and other four-electron oxidoreductases shows some similarity with UDP-D-glucose dehydrogenase and mammalian HMG-CoA reductase. The 500 bp segment upstream from HMG-CoA reductase includes a putative ribosome binding site and a promoter that contains a array of direct- and inverted repeats. The putative promoter also contains sequences homologous to those found in Klebsiella and Pseudomonas promoters. An open reading frame begins immediately following the HMG-GoA reductase gene and extends beyond the fragment sequenced. The potential ribosome binding site for this open reading frame overlaps the end of the HMG-CoA reductase gene and therefore the putative protein may be translationally coupled to HMG-CoA reductase in a mevalonate catabolic operon. Analysis of the amino terminal segment of the putative protein showed that it contains a consensus sequence thought to be involved in metal binding
Purification, characterization, and mechanism of action of hamster HMG-CoA reductase
No full textA method has been developed to purify the catalytic domain of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, E.C. 1.1.1.34) to homogeneity and in high yield. Using the developed purification protocol, together with mutagenesis and in vitro and in vivo complementation experiments, the mammalian enzyme has been determined to be catalytically active as a dimer. The dimeric enzyme has two active sites located at the interface between adjacent polypeptide subunits. Each subunit contributes catalytically essential acidic amino acids to each active site, Glu\sp{558} from one polypeptide and Asp\sp{766} from the adjacent polypeptide. During deacylation of HMG-CoA, it is proposed that Glu\sp{558} functions as a proton donor to His\sp{865}, which then protonates the coenzyme A anion, CoAS\sp-. Protonation of CoAS\sp- is required for the stimulation of the reduction of mevaldehyde by coenzyme A and for enzyme turnover. Asp\sp{766} is proposed to function as a general acid-base calalyst during the overall reaction
Pseudomonas mevalonii HMG-CoA reductase: Transcriptional regulation and identification of a glutamate essential for catalysis
No full textPseudomonas mevalonii HMG-CoA reductase (E.C. 1.1.1.38) is induced in cells grown on mevalonate (J. F. Gill, Jr., M. J. Beach, and V. W. Rodwell, J. Bacteriol. 160, 294-298, 1984), and the mvaA gene, which encodes P. mevalonii HMG-CoA reductase, has been cloned and sequenced (M. J. Beach and V. W. Rodwell, J. Bacteriol. 171, 2994-3001, 1989). Measurements of HMG-CoA reductase activity, protein, and mRNA revealed that induction is controlled at the transcriptional level. The transcription initiation site is 56 bp upstream of the adenosine of the ATG translation start codon of mvaA. The mvaA promoter has 12 and 24 consensus sequences typical of prokaryotic promoters which utilize sigma\sp{54} RNA polymerase holoenzyme and activator proteins for transcription. The cis-acting element which responds to mevalonate was shown, by assays of the expression of mva-lacZ translation fusions and by DNA gel retardation assays, to be on a 36 pb DNA segment 48 pb upstream of the transcription initiation site. From kinetic data, D. Veloso, W. W. Cleland, and J. W. Porter (Biochemistry 20, 887-894, 1981) inferred that an acidic residue functions in catalysis by yeast HMG-CoA reductase. The catalytic domains of 11 HMG-CoA reductases contain 3 conserved acidic residues. These 3 residues of P. mevalonii HMB-CoA reductase were changed to glutamine (E52 and E83), asparagine (D183), or alanine (D183) by site-directed mutagenesis. All four mutant enzymes were then overexpressed, purified, and characterized. V\sb{\rm max} for mutant enzymes E52Q, D183A, and D183N was 15, 69, and 109% that of wild-type HMG-CoA reductase, respectively. Thus, neither Glu\sp{52} nor Asp\sp{183} is a catalytic residue. Mutant enzymes E52Q and D183A had elevated K\sb{\rm m} values for all substrates and impaired ability to bind to coenzyme A or HMG-CoA affinity supports. By contrast, mutant enzyme E83Q had a V\sb{\rm max} value only 0.4% that of wild-type HMG-CoA reductase. This low value appeared to result neither from an altered conformation nor from an impaired inability to bind substrates. For mutant enzyme E83Q, K\sb{\rm m} values for NAD\sp{+}, R,S-mevalonate, and coenzyme A, and chromatography on affinity supports was typical of wild-type enzyme. The data are consistent with residue E83 of P. mevalonii HMG-CoA reductase being the acidic residue functional in catalysis
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