Brassica juncea HMG-CoA synthase: Localization of mRNA and protein

3-Hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) synthase (HMGS; EC 2.3.3.10) synthesizes HMG-CoA, a substrate for mevalonate biosynthesis in the isoprenoid pathway. It catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA and HS-CoA. In Brassica juncea (Indian mustard), HMGS is encoded by four isogenes (BjHMGS1-BjHMGS4). We have already enzymatically characterized recombinant BjHMGS1 expressed in Escherichia coli, and have identified its residues that are significant in catalysis. To further study HMGS mRNA expression that is developmentally regulated in flowers and seedlings, we have examined its mRNA distribution by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). We observed predominant localization of HMGS mRNA in the stigmas and ovules of flower buds and in the piths of seedling hypocotyls. RT-PCR analysis revealed that BjHMGS1 and BjHMGS2 but not BjHMGS3 and BjHMGS4were expressed in floral buds. To investigate the subcellular localization of BjHMGS1, we fused BjHMGS1 translationally in-frame either to the N- or C-terminus of green fluorescent protein (GFP). BjHMGS1-GFP and GFP-BjHMGS1 fusions were used in particle gun bombardment of onion epidermal cells and tobacco BY-2 cells. The GFP-BjHMGS1 construct was also used in agroinfiltration of tobacco leaves. Both GFP-fusion proteins were observed transiently expressed in the cytosol on confocal microscopy of onion epidermal cells, tobacco BY-2 cells, and agroinfiltrated tobacco leaves. Further, subcellular fractionation of total proteins from transgenic plants expressing GFP-BjHMGS1 derived from Agrobacterium-mediated transformation confirmed that BjHMGS1 is a cytosolic enzyme. We suggest that the presence of BjHMGS isoforms is likely related to the specialization of each in different cellular and metabolic processes rather than to a different intracellular compartmentation of the enzyme. © Springer-Verlag 2005.link_to_subscribed_fulltex

Enhancing plant growth, seed yield and sterol content through manipulation of 3-hydroxy-3-methylglutaryl coenzyme A synthase

Poster Session - Biosynthesis: Early Steps and Isoprene Formation: no. P.027Due to the importance of plant isoprenoids, several attempts had been made to achieve some overproduction. Rate-limiting reactions in the MVA pathway like that catalyzed by HMG-CoA reductase were initially considered, also of enzymes downstream. In previous work we had studied the role and kinetics of the second enzyme in the MVA pathway, HMG-CoA synthase in Brassica juncea (BjHMGS1), including the effect of targeted mutation of several amino acid residues and could generate an enzyme with higher catalytic efficiency in vitro. WT BjHMGS1 and BjHMGS1S393A were transformed into Arabidopsis, resulting in positive effects such as an increased resistance to stress. This has been extended to tobacco over-expressing wild-type (OE-wtBjHMGS1) and mutant BjHMGSS359A (OE-S359A), which resulted in higher accumulation of total phytosterols than in the vector-transformed control, with OE-S359A showing some higher sterol increase than OE-wtBjHMGS1. The over-expression of HMGS up-regulated several downstream genes in the isoprenoid pathway and was found to accelerate and promote plant growth, leading to a higher yield of seeds, which both are interesting agricultural traits. The role of HMGSfor plant growth is supported by the existence of natural inhibitors like F-244, efficiently retarding growth of B juncea seedlings and tobacco BY 2 cells

Past achievements, current status and future perspectives of studies on 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) in the mevalonate (MVA) pathway

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Arabidopsis overexpressing wild-type and mutant Brassica juncea HMG-CoA synthase show unregulation of genes in sterol biosynthesis (invited talk)

Session Gen-1: Genetic Engineering for Agriculture Improvemen

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

3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2, 4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS. © 2006 by The National Academy of Sciences of the USA.link_to_OA_fulltex

Enhanced sterol production and stress-tolerance in Arabidopsis overexpressing wild-type and mutant Brassica juncea HMGSI

CSH Asia 2010 - Cold Spring Harbor Asia Conferences: From Plant Biology to Crop Biotechnology, Suzhou, China, 25-29 October 2010