Purification, characterization, and cloning of a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in biosynthesis of terpenoids

Abstract

The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) was purified 3,000-fold from Streptomyces sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 microM for NADPH and 7.7 microM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.

FIG. 1

Electrophoresis of the purified HMG-CoA reductase. Purified HMG-CoA reductase was analyzed by SDS–8 to 25% PAGE (left) and native 8 to 25% PAGE (right). Lanes: 1, SDS-treated enzyme (0.8 μg); 2 and 3, molecular mass standard; 4, native enzyme (1.0 μg). Proteins were stained with Coomassie brilliant blue R-250.

FIG. 2

Double-reciprocal plots for inhibition by pravastatin. HMG-CoA reductase activity of the purified CL190 enzyme was assayed in the presence of 0 μM (●), 1 μM (○), or 10 μM (■) pravastatin as described in Materials and Methods, except that the assay was initiated by adding NADPH. Each point represents the average of two determinations.

FIG. 3

Comparison of amino acid sequences of HMG-CoA reductase. A multiple alignment of the amino acid sequences was determined by using the GENETYX program. Identical amino acids among nine proteins are marked by asterisks and sharps. Dashes indicate gaps introduced for optimization of the alignment. Indicated numbers refer to amino acid positions. CL, Streptomyces sp. strain CL190; Hs, Homo sapiens (SWISS-PROT, P04035); Rn, Rattus norvegicus (SWISS-PROT, P51639); Dm, Drosophila melanogaster (SWISS-PROT, P14773); At, A. thaliana (SWISS-PROT, P14891); Sc, Saccharomyces cerevisiae (SWISS-PROT, P12683); Mj, Methanococcus jannaschii (SWISS-PROT, Q58116); Ss, Sulfolobus solfataricus (DAD, U95360); Hv, Haloferax volcanii (SWISS-PROT, Q59468). I, II, III, and IV indicate proposed binding sites for HMG-CoA (I and II) and NAD(P) (III and IV) (29). Sharps indicate amino acids that have been proposed to function in catalysis (6, 13, 14, 18, 48). Motifs A to G indicate regions in which the sequences were highly conserved. The dotted line over the sequences after motif G indicates putative kinase recognition sequences that are conserved in higher eucaryotes.

FIG. 4

Structures and conserved motifs of the HMG-CoA reductase proteins. Location of conserved motifs I to IV and A to G are indicated by shaded and open boxes, respectively. Pm indicates the HMG-CoA reductase from P. mevalonii. Other abbreviations used are as in Fig. 3.