Expression and Characterization of the HMG-CoA Reductase of the Thermophilic Archaeon Sulfolobus solfataricus

Kim, Dong-Yul; Bochar, Daniel A.; Stauffacher, Cynthia V.; Rodwell, Victor W.

doi:10.1006/prep.1999.1147

Cited by 12 publications

(4 citation statements)

References 27 publications

(69 reference statements)

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“…As shown in Fig. S3A, simvastatin lactone (a statin inhibitor) strongly inhibited mHMGR ( app K i = 2.9 ± 0.8 n m ) similar to the degree of statin inhibition on class‐I HMGRs, such as human HMGR ( app K i = 11 n m for simvastatin) and archaeal HMGRs including S. solfataricus HMGR ( app K i for Lovastatin = 5 n m ) and H. volcanii HMGR ( app K i for Lovastatin lactone = 15 μ m and app K i for Lovastatin free‐acid 15 n m ) . We also tested the effect of simvastatin lactone on the growth of M. thermolithotrophicus (Fig.…”

Section: Resultssupporting

confidence: 53%

Crystal structure of archaeal HMG‐CoA reductase: insights into structural changes of the C‐terminal helix of the class‐I enzyme

et al. 2019

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3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR) catalyses the last step in mevalonate biosynthesis. HMGR is the target of statin inhibitors that regulate cholesterol concentration in human blood. Here, we report the properties and structures of HMGR from an archaeon Methanothermococcus thermolithotrophicus (mHMGR). The structures of the apoenzyme and the NADPH complex are highly similar to those of human HMGR. A notable exception is C‐terminal helix (Lα10‐11) that is straight in both mHMGR structures. This helix is kinked and closes the active site in the human enzyme ternary complex, pointing to a substrate‐induced structural rearrangement of C‐terminal in class‐I HMGRs during the catalytic cycle.

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Section: Resultssupporting

confidence: 53%

Crystal structure of archaeal HMG‐CoA reductase: insights into structural changes of the C‐terminal helix of the class‐I enzyme

et al. 2019

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“…By contrast, genes that encode class II forms of the enzyme are present in some eubacteria and in the archaeon Archaeoglobus fulgidus (41). Previously characterized class I HMG-CoA reductases include those of the archaea Haloferax volcanii (6) and Sulfolobus solfataricus (7,22), the gram-positive eubacterium Streptomyces sp. strain CL190 (40), and numerous eukaryotes.…”

mentioning

confidence: 99%

“…strain CL190 (40), and numerous eukaryotes. Characterized class II HMG-CoA reductases include that from A. fulgidus (23) and the biodegradative Pseudomonas mevalonii enzyme (4,20), whose structure has been determined (24,39) and which converts mevalonate to HMG-CoA, permitting growth on mevalonate (15). No biosynthetic eubacterial class II HMG-CoA reductase has, however, been characterized.…”

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confidence: 99%

Essentiality, Expression, and Characterization of the Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase of Staphylococcus aureus

Wilding¹,

Kim²,

Bryant³

et al. 2000

J Bacteriol

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Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K i of 320 M, over 10 4 higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.

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“…Genes encoding wild-type or mutant HMG-CoA reductases were expressed in E. coli BL21(DE3) cells. The resulting enzymes were then purified by the protocol developed for purification of the wild-type enzyme (22). Table 2 summarizes the purification of the wild-type enzyme and of enzyme L403R/G404R/A406S.…”

Section: Methodsmentioning

confidence: 99%

Engineering of Sulfolobus solfataricus HMG-CoA Reductase to a Form Whose Activity Is Regulated by Phosphorylation and Dephosphorylation

2000

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There are two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase: the class I enzymes of eukaryotes and some archaea, and the class II enzymes of certain eubacteria. The activity of the class I Syrian hamster HMG-CoA reductase is regulated by phosphorylation-dephosphorylation of Ser871. Phosphorylation apparently prevents the active site histidine, His865, from protonating the inhibitory coenzyme A thioanion prior to its release from the enzyme. Structural evidence for this hypothesis is, however, lacking. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus, whose stability recommends it for physical studies, lacks both a phosphoacceptor serine and a protein kinase recognition motif. Consequently, its activity is not regulated by phosphorylation. We therefore employed site-directed mutagenesis to engineer an appropriately located phosphoacceptor serine and cAMP-dependent protein kinase recognition motif. Substitution of serine for Ala406, the apparent cognate of hamster Ser871, and replacement of Leu403 and Gly404 by arginine created S. solfataricus mutant enzyme L403R/G404R/A406S. The general properties of enzyme L403R/G404R/A406S (K(m) values, V(max), optimal pH and temperature) were essentially those of the wild-type enzyme. Exposure of enzyme L403R/G404R/A406S to [gamma-(32)P]ATP and cAMP-dependent protein kinase was accompanied by incorporation of (32)P(i) and by a parallel decrease in catalytic activity. Subsequent treatment with a protein phosphatase released enzyme-bound (32)P(i) and restored activity to pretreatment levels. The regulatory properties of enzyme L403R/G404R/A406S thus match those of the hamster enzyme. Solution of the three-dimensional structures of the phospho and dephospho forms of this mutant enzyme thus should reveal structural features critical for regulation of the activity of a class I HMG-CoA reductase.

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Expression and Characterization of the HMG-CoA Reductase of the Thermophilic Archaeon Sulfolobus solfataricus

Cited by 12 publications

References 27 publications

Crystal structure of archaeal HMG‐CoA reductase: insights into structural changes of the C‐terminal helix of the class‐I enzyme

Crystal structure of archaeal HMG‐CoA reductase: insights into structural changes of the C‐terminal helix of the class‐I enzyme

Essentiality, Expression, and Characterization of the Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase of Staphylococcus aureus

Engineering of Sulfolobus solfataricus HMG-CoA Reductase to a Form Whose Activity Is Regulated by Phosphorylation and Dephosphorylation

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