Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5505690 | Biochemical and Biophysical Research Communications | 2017 | 6 Pages |
Abstract
Amino acid biosynthesis has emerged as a source of new drug targets as many bacterial strains auxotrophic for amino acids fail to proliferate under in vivo conditions. Branch chain amino acids (BCAAs) are important for Mycobacterium tuberculosis (Mtb) survival and strains deficient in their biosynthesis were attenuated for growth in mice. Threonine dehydratase (IlvA) is a pyridoxal-5-phosphate (PLP) dependent enzyme that catalyzes the first step in isoleucine biosynthesis. The MRA_1571 of Mycobacterium tuberculosis H37Ra (Mtb-Ra), annotated to be coding for IlvA, was cloned, expressed and purified. Purified protein was subsequently used for developing enzyme assay and to study its biochemical properties. Also, E. coli BL21 (DE3) IlvA knockout (E. coli-ÎilvA) was developed and genetically complemented with Mtb-Ra ilvA expression construct (pET32a-ilvA) to make complemented E. coli strain (E. coli-ÎilvA + pET32a-ilvA). The E. coli-ÎilvA showed growth failure in minimal medium but growth restoration was observed in E. coli-ÎilvA + pET32a-ilvA. E. coli-ÎilvA growth was also restored in the presence of isoleucine. The IlvA localization studies detected its distribution in cell wall and membrane fractions with relatively minor presence in cytosolic fraction. Maximum IlvA expression was observed at 72 h in wild-type (WT) Mtb-Ra infecting macrophages. Also, Mtb-Ra IlvA knockdown (KD) showed reduced survival in macrophages compared to WT and complemented strain (KDC).
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Authors
Rishabh Sharma, Deepa Keshari, Kumar Sachin Singh, Sudheer Kumar Singh,