The Thermophilic, Homohexameric Aminopeptidase of Borrelia burgdorferi Is a Member of the M29 Family of Metallopeptidases
2005
Bertin, Patrícia B. | Lozzi, Silene P. | Howell, Jerrilyn K. | Restrepo-Cadavid, Glória | Neves, David | Teixeira, Antonio R. L. | de Sousa, Marcelo V. | Norris, Steven J. | Santana, Jaime M.
Proteases are implicated in several aspects of the physiology of microorganisms, as well as in host-pathogen interactions. Aminopeptidases are also emerging as novel drug targets in infectious agents. In this study, we have characterized an aminopeptidase from the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. The aminopeptidolytic activity was identified in cell extracts from B. burgdorferi by using the substrate leucine-7-amido-4-methylcoumarin. A protein displaying this activity was purified from B. burgdorferi by a two-step chromatographic procedure, yielding a [approximately]300-kDa homo-oligomeric enzyme formed by monomers of [approximately]50 kDa. Gel enzymography experiments showed that enzymatic activity depends on the oligomeric structure of the protease but does not involve interchain disulfide bonds. The enzyme was identified by peptide mass fingerprinting as the putative aminopeptidase II of B. burgdorferi, encoded by the gene BB0069. It shares significant identity to members of the M29/T family of metallopeptidase, is sensitive to bestatin, has a neutral pH optimum, and displays maximal activity at 60°C. Its activity is 1.75-fold higher at the temperature of the mammalian host than at that of the insect host of the pathogen. The activity of this thermophilic aminopeptidase of B. burgdorferi (TAP[subscript Bb]) depends on Zn²⁺, and temperatures over 70°C promoted its inactivation through a transition from the hexameric state to the monomeric state. Since B. burgdorferi is deficient in pathways for amino acid synthesis, TAP[subscript Bb] could play a role in supplying required amino acids. Alternatively, the enzyme could be involved in peptide and/or protein processing.
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