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An aminopeptidase nutritionally important to Fusobacterium nucleatum

A. Gunadi

Microbiology Laboratory, Department of Dentistry, The University of Adelaide, North Terrace, Adelaide, South Australia 5005, Australia

Author for correspondence: A. H. Rogers. Tel: + 61 8 8303 5104. Fax: + 61 8 8303 3444.

ABSTRACT

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 pl of 5.1, a pH optimum between 7.5 and 8.0 and, using Leu-Ala as substrate, it gave K_m and V_max values of 0.66 mM and 0.12 µmol 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.

Present address: Universitas Jember, Jalan Kalimantan 1 no. 58, Jember-Jawa Timur 68121, Indonesia.

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