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.
Rogers
A H
AH
Gunadi
A
A
Gully
N J
NJ
Zilm
P S
PS
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003035:Cobalt / Q000494:pharmacology; D016967:Fusobacterium nucleatum / Q000201:enzymology* / Q000502:physiology; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D001426:Bacterial Proteins; D002614:Chelating Agents; D006902:Hydroxymercuribenzoates; D010618:Phenanthrolines; D011480:Protease Inhibitors; D003035:Cobalt; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex