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Acta Biochimica et Biophysica Sinica Advance Access originally published online on March 24, 2009
Acta Biochimica et Biophysica Sinica 2009 41(5):379-388; doi:10.1093/abbs/gmp017
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Characterization of the putative tryptophan synthase β-subunit from Mycobacterium tuberculosis

Hongbo Shen1,{dagger}, Yanping Yang1,{dagger}, Feifei Wang1, Ying Zhang2, Naihao Ye3, Shengfeng Xu1,* and Honghai Wang1,*

1 State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China
2 Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Room E2037, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA
3 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China

* Correspondence address. Tel: +86-21-65643777; Fax: +86-21-65648376; E-mail: hhwang{at}fudan.edu.cn (H.W.); Tel: +86-21-65643777; Fax: +86-21-65648376; E-mail: xushengfeng2001{at}hotmail.com (S.X.)


  Abstract

The increasing emergence of drug-resistant tuberculosis (TB) poses a serious threat to the control of this disease. It is in urgent need to develop new TB drugs. Tryptophan biosynthetic pathway plays an important role in the growth and replication of Mycobacterium tuberculosis (Mtb). The β-subunit of tryptophan synthase (TrpB) catalyzes the last step of the tryptophan biosynthetic pathway, and it might be a potential target for TB drug design. In this study, we overexpressed, purified, and characterized the putative TrpB-encoding gene Rv1612 in Mtb H37Rv. Results showed that Mtb His-TrpB optimal enzymatic activity is at pH 7.8 with 0.15 M Na+ or 0.18 M Mg2+ at 37°C. Structure analysis indicated that Mtb TrpB exhibited a typical β/{alpha} barrel structure. The amino acid residues believed to interact with the enzyme cofactor pyridoxal-5'-phosphate were predicted by homology modeling and structure alignment. The role of these residues in catalytic activity of the Mtb His-TrpB was confirmed by site-directed mutagenesis. These results provided reassuring structural information for drug design based on TrpB.

Keywords    tryptophan synthase; Mycobacterium tuberculosis; enzyme activity; active site; site-directed mutation

Received: December 10, 2008; Accepted: March 3, 2009

{dagger} These authors contributed equally to this work.


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