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Chemical biology of Mycobacterium
tuberculosis: deciphering the role of polyketide synthases in mycobacteria |
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Principal Investigator : Rajesh S Gokhale Project Associate/Assistant Ph D Students Collaborator This laboratory is interested in genome-based
approaches to identify and exploit the microbial metabolic pathways that are
involved in the biosynthesis of various natural products. The present focus is
to understand the importance of various polyketide synthase gene clusters from
Mycobacterium tuberculosis. The genome sequence of M.tuberculosis has revealed
a remarkable array of genes that are homologous to polyketide synthases.
Polyketide synthases (PKSs) are a class of enzymes that are involved in the
biosynthesis of secondary metabolites such as erythromycin, rapamycin,
tetracycline, lovastatin and resveratrol. Our study attempts to understand and
exploit the role of polyketide synthases in the biology of M.tuberculosis.
Previous studies with Actinomycetes PKS systems have shown that genes involved
in the biosynthesis of a particular metabolite are clustered in one region of
the genome. We have therefore decided to dedicate our initial efforts in
studying two large PKS gene clusters that have been identified during the
genome sequencing of M.tuberculosis. A. PKS gene cluster consisting of architecturally
two different types of PKS proteins This gene cluster contains six different open reading
frames (ORFs). Our analysis suggested that there are architecturally at least
two different types of PKS genes in this cluster. Four modular genes, pks7,
pks8, pks17, and pks9 are flanked on either side by two chalcone synthase-like
genes, pks10 and pks11. Such an unusual organization of the gene cluster has
not been reported earlier. Further analyses of their domains suggested that
pks7 is one complete module with all three reductive domains. pks8 in contrast
is only a partial module, as the acyl carrier protein (ACP) domain is absent
from this protein. Remarkably, pks17 complements by providing rest of the
domains. pks9 gene is similar to several chain initiation modules, where
active site cys residue of ketosynthase (KS) domain is mutated to gln (KSQ).
Such modules have been implicated in decarboxylation of the starter units.
Chalcone synthases have been traditionally studied from plant kingdom. These
enzymes were recently characterized in Streptomyces as well. Our approach for studying this PKS gene cluster is
based on the expression of their cognate genes in S.coelicolor and E.coli. We
have been able to produce pks10 and pks11 proteins in E.coli. These proteins
were expressed as hexa-histidine fusion constructs and were purified to
homogeneity using Ni-NTA affinity column. We are presently trying to decipher
the substrate specificity of these proteins. It is known that chalcone
synthase-like proteins use different acyl-coenzyme A as substrates, which
results in the biosynthesis of diverse metabolic products. In order to predict
the substrates for pks10 and pks11, we have initiated a structure-based
modeling approach in collaboration with Dr. Mohanty’s group. The crystal
structures of two chalcone synthase-like proteins and their complexes with
several substrates and inhibitors are available in Protein Data Bank. By using
homology modeling, we are trying to predict the structures along with their
substrate specificity for the mycobacterial chalcone synthases B. Virulent gene cluster containing
ppsA-E genes The ppsA-E gene cluster consists of five modular PKS
genes. These genes are a part of a large gene cluster spanning 50 kb of DNA,
which putatively codes for 16 ORFs. These genes have been implicated in the
biosynthesis of phthiocerol and phenolphthiocerol derivatives, a group of
molecules restricted to eight mycobacterial species, seven of them being
strict or opportunistic pathogens. This cluster consists of (1) PKS genes (ppsA-E),
(2) the FAS gene mycocerosic acid synthase (mas), (3) several acyl-CoA
synthases and (4) number of genes homologous to transporter proteins. We have
carried out a detailed sequence analysis of pps genes to identify various
domains and catalytic sites present in the modular PKS proteins. These
analyses have identified a number of unique features of these modular PKS
proteins. ppsA contains an additional acyl carrier protein (ACP) domain
located at the N-terminus end of the protein. The C-terminal end of ppsE
protein possesses a domain that is homologous to condensation domain of
non-ribosomal polypeptide synthetase (NRPS) proteins. Such domains have not
been typically characterized in a modular PKSs. Based on the modular logic of
the polyketide biosynthesis, combined with detailed analysis of various
domains present in a given module, we are trying to ascertain the structure of
the polyketide product. In order to assist our predictions, we have
constructed a library of sequences of KS and AT domains from different PKSs.
On the basis of sequence homology, molecular modeling and available homologous
crystal structures, we are attempting to predict the amino acid residues that
play a crucial role in determining the substrate specificity. We have cloned number of these genes in E.coli by
shot-gun method. We are presently engineering suitable restriction sites for
cloning and expressing these proteins in E.coli and Streptomyces expression
vectors. Publications Reviews/Proceedings 1. Gokhale RS and Tuteja D (2001) Biochemistry
of polyketide synthases In: Biotechnology (Eds. Reid and Rehm), WILEY-VCH
Verlag GmbH Germany, 10 (in press). |