An aminopeptidase nutritionally important to Fusobacterium nucleatum

The properties of an aminopeptidase (AP) from Fusobacterium nucleatum were studied in view of the fact that this organism, along with other Gram-negative anaerobes involved in periodontal diseases, survives in the subgingival environment by obtaining energy via the fermentation of a small number of peptide-derived amino acids. The AP was found to be cell-associated and was isolated from disrupted chemostat-grown cells. It was purified by (NH4)2SO4 fractionation, two column chromatographic steps and IEF. The enzyme was found to have a molecular mass of 54 kDa, a pI of 5.1, a pH optimum between 7.5 and 8.0 and, using Leu-Ala as substrate, it gave K(m) and Vmax values of 0.66 mM and 0.12 mumol min-1 mg-1, respectively. No complete homology was found between the N-terminal sequence of the first 20 amino acids (MDXKXYVDLKERFLRYVKFN...) and any other published sequence, but residues 8-20 gave a 62% match with residues 9-21 of an AP from Haemophilus influenzae. The enzyme was inactivated by chelating agents, bestatin, p-hydroxymercuribenzoate and some heavy metals. Cobalt ions restored EDTA-inactivated activity but did not reverse inhibition by 1,10-phenanthroline. In addition, bestatin and 1,10-phenanthroline had an inhibitory effect on the batch growth of F. nucleatum in a complex medium in which peptidase activities would be nutritionally essential. No such inhibition was observed in a chemically defined medium in which growth was not dependent upon peptidase activities. The peptidase described in this paper therefore appears to be a cobalt-activated metallo-AP which, together with other peptidases, is considered to be important in the survival of F. nucleatum in the subgingival environment of the mouth.