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 beta-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 Mg(2+) at 37 degrees C. Structure analysis indicated that Mtb TrpB exhibited a typical beta/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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism*
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Binding Sites / genetics
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Catalysis
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Chromatography, High Pressure Liquid
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Dimerization
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Enzyme Stability / drug effects
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Escherichia coli / genetics
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Kinetics
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Magnesium / pharmacology
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Models, Molecular
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Molecular Sequence Data
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Mutation
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Mycobacterium tuberculosis / enzymology*
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Mycobacterium tuberculosis / genetics
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Protein Subunits / chemistry
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Protein Subunits / genetics
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Protein Subunits / metabolism
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Pyridoxal Phosphate / chemistry
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Pyridoxal Phosphate / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Sodium / pharmacology
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Substrate Specificity
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Temperature
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Tryptophan Synthase / chemistry
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Tryptophan Synthase / genetics
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Tryptophan Synthase / metabolism*
Substances
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Bacterial Proteins
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Protein Subunits
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Recombinant Proteins
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Pyridoxal Phosphate
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Sodium
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Tryptophan Synthase
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Magnesium