Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible

The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway. An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases. The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence. The protein is truncated at its 5 end, and shows reduced sequence conservation in this region as compared to other plant sequences. The rice genome contains a small family of HMGR genes. The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants. It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogenMagnaporthe grisea, causal agent of rice blast disease. This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice.     

Cloning of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from terpenoid antibiotic-producing Streptomyces strains

We have isolated a mutant lacking 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity from a terpenoid antibiotic (terpentecin) producer, Streptomyces griseolosporeus MF730-N6, which uses both the mevalonate and nonmevalonate pathways for the formation of isopentenyl diphosphate, by screening terpentecin non-producing mutants. Terpentecin is known to be synthesized via the mevalonate pathway. The gene encoding HMG-CoA reductase (hmgg) was cloned and identified by complementation of the mutant, using a self-cloning system developed in this study for strain MF730-N6. The corresponding hmgs gene for HMG-CoA reductase was also cloned from Streptomyces sp. KO-3988, which produces the terpenoid antibiotic furaquinocin. Sequence analysis of hmgg and hmgs showed that both genes encode polypeptides of 353 amino acids which are 84% identical to each other. A search of protein sequence databases revealed that both gene products were also similar to HMG-CoA reductases from a variety of other organisms, including Streptomyces sp. CL190 (hmgg is 89% and hmgs 85% identical to its CL190 homolog), sea urchin (40.3 and 40.5%), German cockroach (37.6 and 38.4%), and Camptotheca acuminata (39.7 and 40.8%). Received: 17 May 1999 / Accepted: 10 September 1999     

Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases

A cDNA library from freshly isolated protoplasts was differentially screened using cDNAs from mesophyll cells, stressed leaf strips and cell suspension cultures. One of the selected clones, 6P229, turned out to encode a putative polypeptide showing homology to the btuE periplasmic protein of Escherichia coli and to animal selenium-dependent glutathione peroxidases. A major difference was that the putative selenocysteine in the active site was not encoded by the termination codon TGA. The 6P229 gene was found to be expressed in germinating seeds, in apex and in flowers, as well as in stressed tissues. This pattern of expression would be consistent with a key role in cellular metabolism such as defense against oxidative stresses.     

Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat.     

Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in maize tissues

The activity of the enzyme 3-hydroxy-3-methlglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34) is highly expressed in 4-day-old etiolated seedlings of normal (cv. DeKalb XL72AA), dwarf ( d ₅) and albino ( lw ₃) maize ( Zea mays L.). HMGR activity of maize seedlings appeared to be exclusively associated with the microsomal rather than the plastidic fraction of maize cells. Maize tissues with high meristematic activity such as germinating seeds, leaf bases, root tips and the site of origin of lateral roots contained high levels of microsomal HMGR activity. The activity of HMGR extracted from leaf tips of normal, dwarf and albino maize seedlings is regulated by light. Microsomal HMGR activity from leaf tips of 4-day-old maize seedlings was inhibited significantly following exposure to strong light (600 μmol m⁻² s⁻¹) for more than 10 h. By comparison, microsomal HMGR activity from leaf bases and root tips of maize was not inhibited by exposure to strong light. These results suggest that the microsomal HMGR which is highly expressed in maize may be related to sterol biosynthesis and membrane biogenesis rather than plastidic-associated isoprenoid synthesis and that light may regulate HMGR activity indirectly by increasing cell differentiation.     

Multiple phosphorylation of rat liver 3-hydroxy 3-methylglutaryl coenzyme a reductase

Rat liver homogeneous ³²P-labeled hydroxy methylglutaryl coenzyme A reductase, was treated independently with CNBr and trypsin and the resulting [³²P]phosphopeptides were analyzed by disc gel electrophoresis. CNBr treatment produced only one ³²P-fragment of Mr 18,000. The time course of trypsin hydrolysis initially showed the appearance of some phosphopeptides, which were lately converted in two phosphopeptides of low Mr. These results provide direct support for the concept that hydroxy methyl glutaryl coenzyme A reductase kinase solubilized from microsomes phosphorylates only two sites or set of sites in the reductase molecule.     

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm,Pinus sylvestris

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78–82%) than to chloroplastic (GS2) (71–75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.This paper is dedicated to the memory of Dr. Jesús S. Olavarría.     

Isolation and immunological characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) has been isolated from human liver utilizing HMG-CoA affinity chromatography. The apparent monomer molecular weight of purified human HMG-CoA reductase by SDS-gel electrophoresis was 53,000, and the oligomeric molecular weight determined by sucrose density centrifugation was 104,000. A monospecific antibody prepared against rat liver HMG-CoA reductase inhibited the enzymic activity of microsomal and purified human liver enzyme and formed a single immunoprecipitin line by radial immunodiffusion. These results represent the initial isolation and characterization of human liver HMG-CoA reductase.     

Cellular distribution of 3-hydroxy-3-methylglutaryl coenzyme a reductase and mevalonate kinase in leaves of Nepeta cataria

In Nepeta cataria leaf tissue there are two separate activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and mevalonate (MVA) kinase respectively as determined by the use of a 20–45% discontinuous sucrose density gradient. Cell-free extracts of leaf and callus tissue were prepared and HMG-CoA reductase and MVA kinase activities were compared to activities in extracts from porcine livers and yeast autolysates. Callus tissue from N. cataria has only one peak of HMG-CoA reductase and MVA kinase activity located at the top of the sucrose density gradient. Isolated chloroplast from N. cataria leaves have one peak of HMG-CoA reductase and MVA kinase activity, located near the bottom of a sucrose density gradient. MVA kinase activities in porcine livers and yeast autolysate also showed only one activity profile, located at the top of the sucrose gradient. Partial purification of the leaf extract through the use of differential centrifugation, 30–70% ammonium sulfate precipitation and Bio-Gel P-100 column chromatography shows that MVA kinase, 5-phosphomevalonate (MVAP) kinase and 5-pyrophosphomevalonate (MVAPP) decarboxylase activities remain in the same fractions. The extra-chloroplastidic HMG-CoA reductase activity may be separated from MVA kinase activity by differential centrifugation. These results suggest the presence of two HMG-CoA reductase and MVA kinase enzymes in N. cataria leaf tissue—one located in the chloroplast and a second being extra-chloroplastidic.     

Human hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase: evidence for the regulation of enzymic activity by a bicyclic phosphorylation cascade

Microsomal human liver HMG-CoA reductase has been shown to exist in active (dephosphorylated) and inactive (phosphorylated) forms. Microsomal HMG-CoA reductase was inactivated in vitro by ATP-Mg in a time dependent manner; this inactivation was mediated by reductase kinase. Incubation of inactivated enzyme with phosphatase resulted in a time dependent reactivation (dephosphorylation). Polyacrylamide gel electrophoresis of purified HMG-CoA reductase incubated with reductase kinase and radiolabeled ATP revealed that the 32P radioactivity and HMG-CoA reductase enzymic activity were localized in a single electrophoretic position. Partial dephosphorylation of the phosphorylated enzyme was associated with loss of 32P and increase in HMG-CoA reductase activity. Human reductase kinase also exists in active and inactive forms. The active (phosphorylated) form of reductase kinase can be inactivated by incubation with phosphatase. Phosphorylation of inactive reductase kinase with ATP-Mg and a second kinase, reductase kinase kinase, was associated with a parallel increase in the enzymic activity of reductase kinase and the ability to inactivate HMG-CoA reductase. The combined results present initial evidence for the presence of human HMG-CoA reductase and reductase kinase in active and inactive forms, and the in vitro modulation of its enzymic activity by a bicyclic phosphorylation cascade. This bicyclic cascade system may provide a mechanism for short-term regulation of the pathway for cholesterol biosynthesis in man.     

Molecular cloning,characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the first committed step in MVA pathway for isoprenoid biosynthesis in plants. In this study, a full-length cDNA encoding HMGR was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmHMGR (GenBank Accession No.EU680958). The full-length cDNA of SmHMGR was 2,115 bp containing a 1,695 bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyzes revealed that the deduced SmHMGR had extensive homology with other plant HMGRs contained two transmembrane domains and a catalytic domain. Molecular modeling showed that SmHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. Phylogenetic tree analysis indicated that SmHMGR belongs to the plant HMGR super-family and has the closest relationship with HMGR from Picrorhiza kurrooa. Expression pattern analysis implied that SmHMGR expressed highest in root, followed by stem and leaf. The expression of SmHMGR could be up-regulated by salicylic acid (SA) and methyl jasmonate (MeJA), suggesting that SmHMGR was elicitor-responsive. This work will be helpful to understand more about the role of HMGR involved in the tanshinones biosynthesis at the molecular level.     

Proteinase involvement in the solubilization of 3-hydroxy-3-methylglutaryl coenzyme a reductase

This paper demonstrates that a heavy particle fraction, which contains lysosomes, is required for the solubilization of HMG-CoA reductase from rat liver microsomes by the widely-used slow freeze-thaw procedure. This solubilization is effectively inhibited by the proteinase inhibitors, leupeptin and antipain, but not by phenylmethylsulfonyl fluoride, pepstatin A or N-α-p-tosyl-L-lysine methyl ester. These results suggest that a thiol proteinase, possibly derived from lysosomes, is responsible for solubilizing the reductase.     

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

Summary A cDNA encoding -ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned fromCuphea lanceolata. This cDNA of 1276 by codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp -ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the -ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, -ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced inEscherichia coli, was isolated and proved to possess -ketoacyl-ACP reductase activity. Hybridization studies revealed that inC. lanceolata -ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.     

Molecular cloning and characterization of a cDNA encoding nitrate reductase from Chlorella ellipsoidea (Chlorophyta)

Nitrate reductase (NR) genes have beencloned from higher plants, fungi and algae.Based on seven of the amino acid residuesmost strongly conserved between Chlorella vulgaries and Chlamydomonasreinhardtii NR gene, a degenerate primerwas designed. This degenerate primer wasused to amplify the corresponding homologyin Chlorella ellipsoidea. A 3304 bpfull-length cDNA was cloned by rapidamplification of cDNA ends (RACE). Thededuced amino acid sequence of this cDNAhas a high degree of similarity withpreviously identified members of the NRgene. This suggests that the amplified cDNAencodes a functional NR. Northern blotexpression analysis suggests that this geneis strongly induced by nitrate, but isrepressed by ammonium. The nucleotidesequence data reported in this paper willappear in the DDBJ/EMBL/GeneBank databasesunder accession number AY275834.