The Mycobacterium leprae hsp65 Displays Proteolytic Activity. Mutagenesis Studies Indicate That the M. leprae hsp65 Proteolytic Activity Is Catalytically Related to the HslVU Protease

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Abstract
The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (β-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P1, although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T136, K168, and Y264) and the other in the C-domain (T375, K409, and S502). Mutagenesis studies showed that the replacement of K409 by A caused a complete loss of the proteolytic activity, whereas the mutation of K168 to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T375, K409, and S502 at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.