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

TitleBrassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: Expression and characterization of recombinant wild-type and mutant enzymes
Authors
Issue Date2004
PublisherPortland Press Ltd. The Journal's web site is located at http://www.biochemj.org
Citation
Biochemical Journal, 2004, v. 383 n. 3, p. 517-527 How to Cite?
Abstract3-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 HMGSl (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 °C, with an energy of activation of 62.5 J · 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. His 6-BjHMGS1 has an apparent Km-acetyl.CoA of 43 μM and a V max of 0.47 μmol · mg-1 · 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 Vmax, 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.
Persistent Identifierhttp://hdl.handle.net/10722/179341
ISSN
2015 Impact Factor: 3.562
2015 SCImago Journal Rankings: 2.582
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorNagegowda, DAen_US
dc.contributor.authorBach, TJen_US
dc.contributor.authorChye, MLen_US
dc.date.accessioned2012-12-19T09:54:19Z-
dc.date.available2012-12-19T09:54:19Z-
dc.date.issued2004en_US
dc.identifier.citationBiochemical Journal, 2004, v. 383 n. 3, p. 517-527en_US
dc.identifier.issn0264-6021en_US
dc.identifier.urihttp://hdl.handle.net/10722/179341-
dc.description.abstract3-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 HMGSl (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 °C, with an energy of activation of 62.5 J · 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. His 6-BjHMGS1 has an apparent Km-acetyl.CoA of 43 μM and a V max of 0.47 μmol · mg-1 · 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 Vmax, 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.en_US
dc.languageengen_US
dc.publisherPortland Press Ltd. The Journal's web site is located at http://www.biochemj.orgen_US
dc.relation.ispartofBiochemical Journalen_US
dc.subject.meshAcetyl-Coa Hydrolase - Metabolismen_US
dc.subject.meshAmino Acid Sequenceen_US
dc.subject.meshAnimalsen_US
dc.subject.meshArabidopsis Proteins - Chemistryen_US
dc.subject.meshArginine - Geneticsen_US
dc.subject.meshAvian Proteins - Chemistryen_US
dc.subject.meshCations - Metabolismen_US
dc.subject.meshChickens - Geneticsen_US
dc.subject.meshCircular Dichroism - Methodsen_US
dc.subject.meshCockroaches - Geneticsen_US
dc.subject.meshFatty Acids, Unsaturated - Metabolismen_US
dc.subject.meshGene Expression Regulation, Enzymologic - Geneticsen_US
dc.subject.meshHistidine - Biosynthesis - Chemistry - Metabolismen_US
dc.subject.meshHumansen_US
dc.subject.meshHydrogen-Ion Concentrationen_US
dc.subject.meshHydroxymethylglutaryl-Coa Synthase - Biosynthesis - Chemistry - Genetics - Metabolismen_US
dc.subject.meshInsect Proteins - Chemistryen_US
dc.subject.meshKineticsen_US
dc.subject.meshLactones - Metabolismen_US
dc.subject.meshMiceen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshMolecular Weighten_US
dc.subject.meshMustard Plant - Enzymologyen_US
dc.subject.meshMutation - Geneticsen_US
dc.subject.meshPlant Proteins - Chemistry - Geneticsen_US
dc.subject.meshRecombinant Proteins - Metabolismen_US
dc.subject.meshSchizosaccharomyces Pombe Proteins - Chemistryen_US
dc.subject.meshSequence Alignment - Methodsen_US
dc.subject.meshTemperatureen_US
dc.titleBrassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: Expression and characterization of recombinant wild-type and mutant enzymesen_US
dc.typeArticleen_US
dc.identifier.emailChye, ML: mlchye@hkucc.hku.hken_US
dc.identifier.authorityChye, ML=rp00687en_US
dc.description.naturelink_to_OA_fulltexten_US
dc.identifier.doi10.1042/BJ20040721en_US
dc.identifier.pmid15233626en_US
dc.identifier.scopuseid_2-s2.0-9144224870en_US
dc.identifier.hkuros96632-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-9144224870&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume383en_US
dc.identifier.issue3en_US
dc.identifier.spage517en_US
dc.identifier.epage527en_US
dc.identifier.isiWOS:000225279300014-
dc.publisher.placeUnited Kingdomen_US
dc.identifier.scopusauthoridNagegowda, DA=8709830800en_US
dc.identifier.scopusauthoridBach, TJ=24786011400en_US
dc.identifier.scopusauthoridChye, ML=7003905460en_US

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