Cloning and characterization of aspartate-beta-semialdehyde dehydrogenase from Mycobacterium tuberculosis H37 Rv.
OBJECTIVE
To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv.


RESULTS
The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed in Escherichia coli. The ASD enzyme was expressed to levels of 40% but was found to be inactive. Functional ASD was obtained by altering induction and growth conditions and the enzyme was purified to near homogeneity using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The K(m) and V(max) values for the three substrates L-ASA, NADP and Pi, the turnover number and specific activity of the enzyme were determined.


CONCLUSIONS
Functional ASD enzyme of M. tuberculosis was obtained by gene cloning and protein purification using affinity chromatography. The K(cat) and specific activity of the enzyme were 8.49 s(-1) and 13.4 micromol min(-1) microg(-1) respectively.


CONCLUSIONS
The ASD enzyme is a validated drug target. We characterized this enzyme from M. tuberculosis and future work would focus on deducing the three-dimensional structure of the enzyme and design of inhibitors, which could be used as drugs against TB.
Shafiani
S
S
Microbial Biotechnology Laboratory, Department of Biotechnology, Panjab University, Chandigarh, Punjab 160014, India.
Sharma
P
P
Vohra
R M
RM
Tewari
R
R
D001223:Aspartate-Semialdehyde Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D016415:Sequence Alignment
D001223:Aspartate-Semialdehyde Dehydrogenase