Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes.
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.
Nagegowda
Dinesh A
DA
Department of Botany, The University of Hong Kong, Pokfulam, Hong Kong, China.
Bach
Thomas J
TJ
Chye
Mee-Len
ML
D000104:Acetyl-CoA Hydrolase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry; D001120:Arginine / Q000235:genetics; D030161:Avian Proteins / Q000737:chemistry; D002412:Cations / Q000378:metabolism; D002645:Chickens / Q000235:genetics; D002942:Circular Dichroism / Q000379:methods; D003058:Cockroaches / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006639:Histidine / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009149:Mustard Plant / Q000201:enzymology*; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry; D016415:Sequence Alignment / Q000379:methods; D013696:Temperature
D029681:Arabidopsis Proteins; D030161:Avian Proteins; D002412:Cations; D005231:Fatty Acids, Unsaturated; D019476:Insect Proteins; D007783:Lactones; D010940:Plant Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C054282:antibiotic 1233A; D006639:Histidine; D001120:Arginine; D006904:Hydroxymethylglutaryl-CoA Synthase; D000104:Acetyl-CoA Hydrolase