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 Cram-negative anaerobes involved in periodontal diseases, survives in the subgingival environment by obtaining energy via the fermentation o f a small number of peptide-derived amino acids. The AP was found to b e cell-associated and was isolated from disrupted chemostat-grown cell s. It was purified by (NH4)SO4 fractionation, two column chromatograph ic steps and IEF. The enzyme was found to have a molecular mass of 54 kDa, a pr 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 mu mol min(-1) mg(-1), respectively. No complete homology was found between t he N-terminal sequence of the first 20 amino acids (MDXKXYVDLKERFLRYVK FN...) and any other published sequence, but residues 8-20 gave a 62% match with residues 9-21 of an AP from Haemophilus influenzae. The enz yme was inactivated by chelating agents, bestatin, p-hydroxymercuriben zoate and some heavy metals. Cobalt ions restored EDTA-inactivated act ivity but did not reverse inhibition by 1,10-phenanthroline. In additi on, bestatin and 1,10-phenanthroline had an inhibitory effect on the b atch growth of F. nucleatum in a complex medium in which peptidase act ivities would be nutritionally essential. No such inhibition was obser ved in a chemically defined medium in which growth was not dependent u pon peptidase activities. The peptidase described in this paper theref ore appears to be a cobalt-activated metallo-AP which, together with o ther peptidases, is considered to be important in the survival of F. n ucleatum in the subgingival environment of the mouth.