Cutinase-like proteins of Mycobacterium tuberculosis: characterization of their variable enzymatic functions and active site identification.
Discovery and characterization of novel secreted enzymes of Mycobacterium tuberculosis are important for understanding the pathogenesis of one of the most important human bacterial pathogens. The proteome of M. tuberculosis contains over 400 potentially secreted proteins, the majority of which are uncharacterized. A family of seven cutinase-like proteins (CULPs) was identified by bioinformatic analysis, expressed and purified from Escherichia coli, and characterized in terms of their enzymatic activities. These studies revealed a functional diversity of enzyme classes based on differential preferences for substrate chain length. One member, Culp1, exhibited strong esterase activity, 40-fold higher than that of Culp6, which had strong activity as a lipase. Another, Culp4, performed moderately as an esterase and weakly as a lipase. Culp6 lipase activity was optimal above pH 7.0, and fully maintained to pH 8.5. None of the CULP members exhibited cutinase activity. Site-directed mutagenesis of each residue of the putative catalytic triad in Culp6 confirmed that each was essential for activity toward all fatty acid chain lengths of nitrophenyl esters and lipolytic function. Culp1 and Culp2 were present only in culture supernatants of M. tuberculosis, while Culp6, which is putatively essential for mycobacterial growth, was retained in the cell wall, suggesting the proteins play distinct roles in mycobacterial biology.
West
Nicholas P
NP
Mycobacterial Research Program, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag 6, Newtown, NSW, 2042, Australia. n.west@centenary.org.au
Chow
Frances M E
FM
Randall
Elizabeth J
EJ
Wu
Jing
J
Chen
Jian
J
Ribeiro
Jose M C
JM
Britton
Warwick J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002800:Cholinesterase Inhibitors / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010261:Paraoxon / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D002800:Cholinesterase Inhibitors; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D010261:Paraoxon