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

Abstract

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.

Structural basis for the design of potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases

“…BjHMGS1 (GenBank accession no. AAF69804) was cloned, expressed, and purified as described (11). The N-terminal hexahistidine tag was cleaved by thrombin digestion during overnight dialysis against 100 mM NaCl͞25 mM Hepes (pH 7.5) at 4°C.…”

“…Recently, detailed kinetic studies of wild-type and mutant forms of one of the four B. juncea HMGS (BjHMGS), BjHMGS1, demonstrated that (E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid (F-244), alternatively known as 1233A, L-659,699, or hymeglusin, acts as a potent inhibitor with an IC 50 of 35 nM (8)(9)(10)(11)(12). F-244 is a polyketide from Fusarium sp.…”

A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia

“…HMGR catalyzes the key regulatory step of the pathway in plants and in mammals. In addition, HMGS activity has been positively correlated with rubber production in Hevea brasiliensis, suggesting a regulatory role for this enzyme as well (Suvachittanont and Wititsuwannakul 1995;Nagegowda et al 2004). It has long been assumed that the DXP and MVA pathways are independently regulated.…”

Molecular cloning and expression analysis of a new putative gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Salvia miltiorrhiza

“…The 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS, EC 2.3.3.10, formerly EC 4.1.3.5) catalyses the condensation of acetyl-CoA with acetoacetyl-CoA to form HMG-CoA in MVA pathway (Nagegowda et al 2004;Kai et al 2006). The HMG-CoA is then converted by HMGCoA reductase (HMGR) to yield MVA which is subsequently converted to IPP, the universal precursor for the synthesis of isoprenoids, including tanshinones.…”