Academic Projects:
2. Structure function analysis of M. tuberculosis redox enzymes
(Collaborator: Abhijit A. Sardesai, Anil Tyagi)
Redox
processes play an important role in the physiology of M. tuberculosis. Since, M.
tuberculosis spend substantial amount of time in the phagosomal
milieu of host macrophages, it is believed that modulation of the redox reactions might help M. tuberculosis survive the hostile environment therein. We are
interested in the structure and biochemical characterization of redox reactions in M.tuberculosis, with the hope that this will offer useful
insights into the natural resistance of Mycobacteria
towards oxidative killing. Towards achieving this objective, we have undertaken
analysis of M. tuberculosis thioredoxins, alkylhydroperoxidase
and inositol-1-phosphate synthase.
The thioredoxin system
in prokaryotes consists of thioredoxins, thioredoxin reductase and NADPH.
Using electrons from the cellular pool of NADPH, the thioredoxins
act as the major reductants of cellular substrates,
transferring electrons via the thioredoxin reductase. The other major redox
buffer in prokaryotes is glutathione, which is absent in M. tuberculosis. However, M. tuberculosis possess
equivalent mycothiol system, which might buffer redox equilibria. According to the originally annotated genome
sequence, there are three thioredoxins and one
thioredoxin reductase present in M. tuberculosis. Our
characterization has revealed that atleast one of the
three thioredoxins is a pseudo gene, while the redox potential of the other two thioredoxins
is slightly lower than their E. coli
homologues.
The only gene that
appears to be induced by peroxides in M.
tuberculosis is the katG
gene encoding the catalase peroxidase. This is because of the canonical regulator of
oxidative stress-oxyR appears to be mutated in M. tuberculosis. In the isoniazid resistant bacilli, the katG
gene accumulates mutations, thereby potentially rendering oxidative response
ineffective. In these bacilli, the ahpC gene has been observed to be overexpressed. Our characterization of AhpC
has revealed that it undergoes interesting conformational and quaternary
structural changes during catalysis. It
appears to shuttle between
dimeric and decameric
states during reaction cycle. The dimers within the decamers are held
together by ionic forces.
·
Conformational
flexibility of M. tuberculosis Thioredoxin reductase:
Crystal Structure and Normal Mode Analysis (PDF)
Mohd. Akif,
K. Suhre, C. Verma and S.
C. Mande
Acta crystallogr. (2005) D61, 1603- 1611.
·
Expression,
purification, crystallization and preliminary X-ray crystallographic studies of
Mycobacterium tuberculosis thioredoxin reductase
(PDF)
Mohd. Akif,
R. Chauhan and S. C. Mande
Acta Crystallogr D (2004) 60, 777-779
·
Site directed
mutagenesis reveals a novel catalytic mechanism of Mycobacterium
tuberculosis Alkylhydroperoxidase C (PDF)
R. Chauhan
and S. C. Mande
Biochem J. (2002) 367, 255- 261.
·
Characterization
of the Mycobacterium tuberculosis H37Rv alkyl hydroperoxidase
AhpC points to the importance of ionic interactions
in oligomerization and activity (PDF)
R. Chauhan
and S. C. Mande
Biochem J. (2001) 354,
209- 215.
·
Complex evolution
of the inositol-1-phosphate synthase gene among archaea and eubacteria (PDF)
N. Bachhawat
and S. C. Mande
Trends Genet. (2000) 16,
111- 113.
·
Identification of
the ino1 gene of Mycobacterium tuberculosis H37Rv reveals a novel
class of inositol-1-phosphate synthase enzyme (PDF)
N. Bachhawat
and S. C. Mande.
J. Mol. Biol. (1999) 291, 531-536.
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