title: The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 proteolytic activity is catalytically related to the HslVU protease.
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 (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), 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 (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) 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.
authors.affiliation: Center for Applied Toxinology, Butantan Institute, CAT/CEPID, Rua Murajuba 125, São Paulo, SP 05467-010, Brazil.
authors.forename: Fernanda C V
authors.initials: FC
authors.lastname: Portaro
authors.forename: Mirian A F
authors.initials: MA
authors.lastname: Hayashi
authors.forename: Luciana J
authors.initials: LJ
authors.lastname: De Arauz
authors.forename: Mario S
authors.initials: MS
authors.lastname: Palma
authors.forename: Marina T
authors.initials: MT
authors.lastname: Assakura
authors.forename: Célio L
authors.initials: CL
authors.lastname: Silva
authors.forename: Antonio C M
authors.initials: AC
authors.lastname: de Camargo
mesh_terms: D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006360:Heat-Shock Proteins; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009166:Mycobacterium leprae / Q000201:enzymology* / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002364:Caseins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C074119:heat-shock protein 65, Mycobacterium; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D000251:Adenosine Triphosphatases; D018833:Chaperonins