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The thermophilic, homohexameric aminopeptidase of Borrelia burgdorferi is a member of the M29 family of metallopeptidases.

ABSTRACT: 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 degrees 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(Bb)) depends on Zn2+, and temperatures over 70 degrees 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(Bb) could play a role in supplying required amino acids. Alternatively, the enzyme could be involved in peptide and/or protein processing.

SUBMITTER: Bertin PB

PROVIDER: S-EPMC1087410 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature

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The thermophilic, homohexameric aminopeptidase of Borrelia burgdorferi is a member of the M29 family of metallopeptidases.

Bertin Patrícia B PB Lozzi Silene P SP Howell Jerrilyn K JK Restrepo-Cadavid Glória G Neves David D Teixeira Antonio R L AR de Sousa Marcelo V MV Norris Steven J SJ Santana Jaime M JM

Infection and immunity 20050401 4

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 ...[more]

PMID: 15784569

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Project description:The ferric uptake regulator (Fur) family of DNA-binding proteins represses and/or activates gene transcription via divalent metal ion-dependent signal sensing. The Borrelia burgdorferi Fur homologue, also known as Borrelia oxidative stress regulator (BosR), promotes spirochetal adaptation to the mammalian host by directly repressing the lipoproteins required for tick colonization and indirectly activating those required for establishing infection in the mammal. Here, we examined whether the DNA-binding activity of BosR was regulated by any of the four most prevalent transition metal ions in B. burgdorferi, Mn, Fe, Cu, and Zn. Our data indicated that in addition to a structural site occupied by Zn(II), BosR had two regulatory sites that could be occupied by Zn(II), Fe(II), or Cu(II) but not by Mn(II). While Fe(II) had no effect, Cu(II) and Zn(II) had a dose-dependent inhibitory effect on the BosR DNA-binding activity. Competition experiments indicated that Cu(II) had a higher affinity for BosR than Zn(II) or Fe(II). A BosR deficiency in B. burgdorferi resulted in a significant increase in the Cu level but no significant change in the levels of Mn, Fe, or Zn. These data suggest that Cu regulates BosR activity, and BosR in turn regulates Cu homeostasis in B. burgdorferi While this regulatory paradigm is characteristic of the Fur family, BosR is the first one shown to be responsive to Cu(II), which may be an adaptation to the potentially high level of Cu present in the Lyme disease spirochete.IMPORTANCE Transition metal ions serve an essential role in the metabolism of all living organisms. Members of the ferric uptake regulator (Fur) family play critical roles in regulating the cellular homeostasis of transition metals in diverse bacteria, and their DNA-binding activity is often regulated by coordination of the cognate divalent metal ions. To date, regulators with metal ion specificity to Fe(II), Mn(II), Zn(II), and Ni(II) have all been described. In this study, we demonstrate that BosR, the sole Fur homologue in Borrelia burgdorferi, is responsive to Cu(II) and regulates Cu homeostasis in this bacterium, which may be an adaption to potentially Cu-rich milieu in the Lyme disease spirochete. This study has expanded the repertoire of the Fur family's metal ion specificity.

Dataset Information

The thermophilic, homohexameric aminopeptidase of Borrelia burgdorferi is a member of the M29 family of metallopeptidases.

Publications

The thermophilic, homohexameric aminopeptidase of Borrelia burgdorferi is a member of the M29 family of metallopeptidases.

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