Abstract
The PE and PPE multigene families, first discovered during the sequencing of M. tuberculosis H37Rv genome are responsible for antigenic variation and have been shown to induce increased humoral and cell mediated immune response in the host. Using the bioinformatics tools, we had earlier reported that the 225 amino acid residue PE-PPE domain (Pfam: PF08237) common to some PE and PPE proteins has a “serine α/β hydrolase” fold and conserved Ser, Asp and His catalytic triad characteristic of lipase, esterase and cutinase activities. In order to prove experimentally that PE-PPE domain is indeed a serine hydrolase, we have cloned the full-length Rv1430 and its PE-PPE domain into pET-28a vector, expressed the proteins in E. coli and purified to homogeneity. The activity assays of both purified proteins were carried out using p-nitrophenyl esters of aliphatic carboxylic acids with varying chain length (C2–C16) to study the substrate specificity. To characterize the active site of the PE-PPE domain, we mutated the Ser199 to Ala. The activity of the protein in the presence of serine protease inhibitor- PMSF and the mutant protein were measured. Our results reveal that Rv1430 and its PE-PPE domain possess esterase activity and hydrolyse short to medium chain fatty acid esters with the highest specific activity for pNPC6 at 37°C, 38°C and pH 7.0, 8.0. The details of this work and the observed results are reported in this manuscript.
Highlights
The complete genome of the Mycobacterium tuberculosis H37Rv strain comprises about 4000 genes, of which 250 genes are involved in fatty acid metabolism [1]
The full-length Rv1430, PE-Pro–Glu (PE) and Pro–Pro–Glu (PPE) domain (144–369 amino acid residues) and mutant PE-PPE domain were cloned in pET-28a vector and the presence of the inserts was confirmed by restriction enzyme digestion
Our previous computational studies have shown that some PE and PPE proteins hitherto not studied comprise a conserved 225 amino acid PE-PPE domain, this domain would fold into a serine a/b hydrolase architecture and would have esterase, lipase or cutinase activity
Summary
The complete genome of the Mycobacterium tuberculosis H37Rv strain comprises about 4000 genes, of which 250 genes are involved in fatty acid metabolism [1]. Apart from the expected richness of genes involved in fatty acid metabolism, two novel gene families, PE and PPE were identified in H37Rv genome encompassing 10% of coding regions [1]. These two large unrelated families of acidic, glycine-rich proteins are often based on multiple copies of the polymorphic GC-rich repetitive sequences (PGRSs), and major polymorphic tandem repeats (MPTRs). The PE and PPE proteins have the characteristic 110 amino acid and 180 amino acid conserved domains towards the N-terminus, respectively [1,3]
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