Expression, purification, characteristics and homology modeling of the HMGS from Streptococcus pneumoniae.
- Author:
Ya-Li BEN
1
;
Gu-Zhen CUI
;
Chen LI
;
Rui HAN
;
Jie ZHANG
;
Qing-Ye ZHANG
;
Jian WAN
;
De-Li LIU
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Gene Expression Regulation, Bacterial; physiology; Hydroxymethylglutaryl-CoA Synthase; chemistry; genetics; metabolism; Models, Molecular; Molecular Sequence Data; Mutation; Protein Conformation; Streptococcus pneumoniae; enzymology; genetics
- From: Biomedical and Environmental Sciences 2009;22(3):229-236
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo understand the molecular basis for a potential reaction mechanism and develop novel antibiotics with homology modeling for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (HMGS).
METHODSThe genetic engineering technology and the composer module of SYBYL7.0 program were used, while the HMGS three-dimensional structure was analyzed by homology modeling.
RESULTSThe mvaS gene was cloned from Streptococcus pneumoniae and overexpressed in Escherichia coli from a pET28 vector. The expressed enzyme (about 46 kDa) was purified by affinity chromatography with a specific activity of 3.24 micromol/min/mg. Optimal conditions were pH 9.75 and 10 mmol/L MgCl2 at 37 degrees C. The V(max) and K(m) were 4.69 micromol/min/mg and 213 micromol/L respectively. The 3D model of S. pneumoniae HMGS was established based on structure template of HMGS of Enterococcus faecalis.
CONCLUSIONThe structure of HMGS will facilitate the structure-based design of alternative drugs to cholesterol-lowering therapies or to novel antibiotics to the Gram-positive cocci, whereas the recombinant HMGS will prove useful for drug development against a different enzyme in the mevalonate pathway.