Yeast sequencing reports. Molecular cloning and sequencing of the <i>hcs</i> gene, which encodes 3‐hydroxy‐3‐methylglutaryl coenzyme a synthase of <i>Schizosaccharomyces pombe</i>

Summary 3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is an enzyme in mevalonate biosynthesis. In plants, investigations have focused on HMG CoA reductase (HMGR) and less is known of the preceding enzyme, HMGS. To understand the regulation of HMGS, we have isolated a Brassica juncea cDNA encoding HMGS, BjHMGS1, for use as a hybridization probe in Northern blot analyses. BjHMGS is expressed in all plant organs and shows developmental regulation in¯ower, seed and seedling, with highest expression in early development. In seedlings, expression is highest in young hypocotyls and is induced during the greening of etiolated cotyledons. BjHMGS is down-regulated by abscisic acid, osmotic stress and dehydration, the effects of which arrested seedling growth. Thus BjHMGS expression shows correlation with rapid cell division and growth, like HMGR. This is not unexpected, as mevalonate is the precursor to many essential isoprenoid compounds, including sterols for membrane biogenesis. Wounding, methyl jasmonate or salicylic acid induce BjHMGS expression, suggesting that, like HMGR, HMGS is involved in defence. As in animals, coordinated regulation of HMGS with HMGR occurred in B. juncea upon germination and in response to salicylic acid. HMGS assays con®rmed that Escherichia coliexpressed recombinant BjHMGS1 shows HMGS activity that is inhibited by F244, a speci®c inhibitor of HMGS. Southern blot analysis revealed gene families encoding HMGS in Brassica species and a summation of homologous genes in the fusion amphidiploid genome of B. juncea, a bi-parental species derived from diploids B. nigra and B. campestris.

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“…1992); Bg , Blattella germanica (Martinez‐Gonzalez et al . 1993) and Sp , Schizosaccharomyces pombe (Katayama et al . 1995).…”

Section: Resultsmentioning

confidence: 99%

Expression of Brassica juncea 3‐hydroxy‐3‐methylglutaryl CoA synthase is developmentally regulated and stress‐responsive

2000

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“…hmgl + protein levels were elevated when HMG-CoA synthase was overproduced HMG-CoA synthase catalyses the synthesis of HMG-CoA from acetoacetyl-CoA (Goldstein and Brown, 1990), producing the substrate of HMG-CoA reductase. We expressed HMG-CoA synthase under the control of the constitutive adh+ promoter in the plasmid, pSOl (Katayama et al, 1996). We found that overexpression of HMG-CoA synthase resulted in an approximately 1.6-fold increase of native levels of hmgl+ protein ( Figure 11).…”

Section: The Hmgl + Protein Was An Integral Membrane Proteinmentioning

confidence: 98%

Molecular, functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

Lum

Edwards

Wright

1996

Yeast

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The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG‐CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG‐CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG‐CoA reductase gene. The presence of a single HMG‐CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed ‘feed‐forward’ regulation was observed in which elevated levels of HMG‐CoA synthase, the enzyme catalysing the synthesis of the HMG‐CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG‐CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post‐divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG‐CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG‐CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG‐CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.

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“…BjHMGS1 from B. juncea (Bj) [33]; HMGS from A. thaliana (At) [30]; P. sylvestris (Ps) [31]; Homo sapiens (Hs) [41]; Mus musculus (Mm) [42]; Gallus gallus (Gg) [43]; B. germanica (Bg) [44] and Schizosaccharomyces pombe (Sp) [45]. Amino acid residues selected for in vitro mutagenesis are in bold.…”

Section: Figure 4 Alignment Of Deduced Amino Acid Sequences For Hmgs mentioning

confidence: 99%

Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes

Nagegowda

Bach

Chye

2004

Biochemical Journal

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3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS; EC 2.3.3.10) is the second enzyme in the cytoplasmic mevalonate pathway of isoprenoid biosynthesis, and catalyses the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA. In this study, we have first characterized in detail a plant HMGS, Brassica juncea HMGS1 (BjHMGS1), as a His6-tagged protein from Escherichia coli. Native gel electrophoresis analysis showed that the enzyme behaves as a homodimer with a calculated mass of 105.8 kDa. It is activated by 5 mM dithioerythreitol and is inhibited by F-244 which is specific for HMGS enzymes. It has a pH optimum of 8.5 and a temperature optimum of 35 degrees C, with an energy of activation of 62.5 J x mol(-1). Unlike cytosolic HMGS from chicken and cockroach, cations like Mg2+, Mn2+, Zn2+ and Co2+ did not stimulate His6-BjHMGS1 activity in vitro; instead all except Mg2+ were inhibitory. His6-BjHMGS1 has an apparent K(m-acetyl-CoA) of 43 microM and a V(max) of 0.47 micromol x mg(-1) x min(-1), and was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA). Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased V(max), indicating some involvement of these residues in catalytic capacity. Unlike His6-BjHMGS1 and its soluble purified mutant derivatives, the H188N mutant did not display substrate inhibition by AcAc-CoA. Substitution S359A resulted in a 10-fold increased specific activity. Based on these kinetic analyses, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that His-188 is involved in conferring substrate inhibition on His6-BjHMGS1. Substitution of an aminoacyl residue resulting in loss of substrate inhibition has never been previously reported for any HMGS.

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Yeast sequencing reports. Molecular cloning and sequencing of the hcs gene, which encodes 3‐hydroxy‐3‐methylglutaryl coenzyme a synthase of Schizosaccharomyces pombe

Cited by 13 publications

References 15 publications

Expression of Brassica juncea 3‐hydroxy‐3‐methylglutaryl CoA synthase is developmentally regulated and stress‐responsive

Expression of Brassica juncea 3‐hydroxy‐3‐methylglutaryl CoA synthase is developmentally regulated and stress‐responsive

Molecular, functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes

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