The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis.
Mycobacterium tuberculosis is an important human pathogen and has developed sophisticated mechanisms to evade the host immune system. These could involve the use of cyclic nucleotide-dependent signaling systems, since the M. tuberculosis genome encodes a large number of functional adenylyl cyclases. Using bioinformatic approaches, we identify, clone, and biochemically characterize the Rv0805 gene product, the first cyclic nucleotide phosphodiesterase identified in M. tuberculosis and a homologue of the cAMP phosphodiesterase present in Escherichia coli (cpdA). The Rv0805 gene product, a class III phosphodiesterase, is a member of the metallophosphoesterase family, and computational modeling and mutational analyses indicate that the protein possesses interesting properties not reported earlier in this class of enzymes. Mutational analysis of critical histidine and aspartate residues predicted to be essential for metal coordination reduced catalytic activity by 90-50%, and several mutant proteins showed sigmoidal kinetics with respect to Mn in contrast to the wild-type enzyme. Mutation of an asparagine residue in the GNHD motif that is conserved throughout the metallophosphoesterase enzymes almost completely abolished catalytic activity, and these studies therefore represent the first mutational analysis of this class of phosphodiesterases. The Rv0805 protein hydrolyzes cAMP and cGMP in vitro, and overexpression in Mycobacterium smegmatis and E. coli reduces intracellular cAMP levels. The presence of an orthologue of Rv0805 in Mycobacterium leprae suggests that the Rv0805 protein could have an important role to play in regulating cAMP levels in these bacteria and adds an additional level of complexity to cyclic nucleotide signaling in this organism.
Shenoy
Avinash R
AR
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Sreenath
Nandini
N
Podobnik
Marjetka
M
Kovacevic
Miroslav
M
Visweswariah
Sandhya S
SS
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019295:Computational Biology; D000242:Cyclic AMP / Q000378:metabolism; D004252:DNA Mutational Analysis; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D004047:Diethyl Pyrocarbonate