Poster: Late and Moved Abstracts
Abs #
1406: Characterization of Brassica juncea HMG-CoA synthase 1
|
|
Presenter: |
Nagegowda, Dinesh A, dinu33@yahoo.com | Authors | Nagegowda, Dinesh A (A) Bach, Thomas J (B) Chye, Mee-Len (A) | | Affiliations: |
(A): Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong
(B): CNRS (UPR 2357), Institut de Biologie Moleculaire des Plantes, Universite Louis Pasteur, F-67083 Strasbourg, France
| |
|
3-Hydroxy-3-methylglutaryl-coenzyme-A synthase (HMGS), the second enzyme involved in the cytoplasmic mevalonate pathway, catalyzes the functionally irreversible condensation of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. B. juncea has four isogenes encoding HMGS. BjHMGS expression is stress responsive, developmentally regulated and shows coordinated expression with HMGR (Alex et al., 2000. Plant J. 22: 415-426). These findings led us to further characterize BjHMGS1 at the protein level. BjHMGS1 with an N-terminal histidine tag was expressed in E. coli and purified by affinity chromatography on Ni2+-agarose to apparent homogeneity and used for the kinetic studies. BjHMGS1 is inhibited by both substrate and product. Such properties could have an important function in the regulation of the mevalonate pathway, for instance when the sequel enzyme HMGR is inhibited or down-regulated. Piling up substrates and products would immediately diminish HMGS activity in vivo and thereby avoid the risk to accumulate potentially toxic intermediates. In vitro, BjHMGS1 exhibits a pH optimum between pH 7.5 and 8.5, a temperature optimum between 30 and 40oC and is activated by 5 mM DTE, and it is inhibited by F244, specific for HMGS enzymes. Among a variety of cations tested, none, especially Mg++, exerted any stimulating effect on BjHMGS1 activity at 0.5 mM to 10 mM concentration, whereas Mn++, Zn++ and Co++ were inhibitory. Single amino acid substitutions for instance Y35A, K90A and H188N completely knocked out the activity, whereas C212A showed more than 50% reduction in the specific activity when compared to wild type BjHMGS1, suggesting a functional role of these conserved amino acids in enzymatic catalysis.
|
|
