Isolation and characterization of a thermostable aminopeptidase(aminopeptidase T) from Thermus aquaticus YT-1, an extremely thermophilic bacterium.

Minagawa, Etsuo; Kaminogawa, Shuichi; Matsuzawa, Hiroshi; Ohta, Takao; Yamauchi, Kunio

doi:10.1271/bbb1961.52.1755

Cited by 21 publications

(12 citation statements)

References 5 publications

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“…In addition to considerable sequence identity with members of the M29 family, TAP Bb is thermophilic. Its optimal temperature for activity was observed at 60°C, identical to that for the aminopeptidases of G. stearothermophilus and S. thermophilus (3,14) and similar to that determined for T. aquaticus aminopeptidase (70°C) (35). In contrast to these microorganisms, B. burgdorferi is not thermophilic.…”

Section: Discussionsupporting

confidence: 68%

“…Although metalloaminopeptidases exhibit a broad range of metal ion dependence, Zn 2ϩ is the most frequently associated cation (42). Members of the M29 family from T. aquaticus and G. stearothermophilus have Co 2ϩ and Mg 2ϩ as cofactors (35,52). The cofactor for another characterized member of the M29 family, PepS, is unknown; however, it is unlikely to be Zn 2ϩ or Co 2ϩ , since low concentrations of these cations inactivate the enzyme (14).…”

Section: Discussionmentioning

confidence: 99%

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The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

Bertin¹,

Lozzi²,

Howell³

et al. 2005

Infect Immun

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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 twostep chromatographic procedure, yielding a ϳ300-kDa homo-oligomeric enzyme formed by monomers of ϳ50 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°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 Zn 2؉ , and temperatures over 70°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.

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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

Bertin¹,

Lozzi²,

Howell³

et al. 2005

Infect Immun

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“…5c). In general, the optimal temperature of thermo-stable aminopeptidase is between 65 and 100°C [8,14,15,17,[28][29][30][31], whereas the optimal temperature of common aminopeptidase is between 30 and 60°C [32][33][34]. Half of the activity can be remained after incubation at 80°C for 119 min.…”

Section: Discussionmentioning

confidence: 99%

A thermo‐stable lysine aminopeptidase from Pseudomonas aeruginosa: Isolation, purification, characterization, and sequence analysis

Zhou

et al. 2014

J. Basic Microbiol.

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Pseudomonas aeruginosa NJ-814, isolated from garden soil, produced an extracellular aminopeptidase that was purified using ammonium sulfate precipitation and ion exchange chromatography. The purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Mr value of the enzyme was estimated to be 55 kDa. The purified enzyme shows maximum activity at pH 9.0 and 80 °C. It exhibits high thermo-stability. Half of the activity can remain after incubation at 80 °C for 119 min. It is stable within pH range of 7.5-10.5. It is strongly activated by Co(2+) and inhibited by Fe(2+) , Cu(2+) , Ni(2+) , Zn(2+) , and ethylene diamine tetraacetic acid (EDTA). The specificity of the enzyme was investigated. Within several aminoacyl-p-nitroanilines (AA-pNA), Lys-pNA is proven to be the optimal substrate. The Michaelis-Menten constant (Km ) of the enzyme for Lys-pNA and Leu-pNA were 2.32 and 9.41 mM, respectively. Peptide map fingerprinting shows that the sequence of the enzyme is highly similar to aminopeptidase Y from P. aeruginosa 18A. It can be speculated that this enzyme is a Zn(2+) -dependent enzyme and contains two zinc ions in its active site.

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“…The highly similar metallopeptidase AmpT from Thermus aquaticus (a) dimerizes in solution, (b) acts as an aminopeptidase and (c) requires metal cations, ideally Co 2C , for activity. 1,2 The Bacillus stearothermophilus AmpT homologue binds 2.2 cobalt ions per protomer according to equilibrium dialysis, 3 and the Staphylococcus aureus enzyme, known as AmpS, has two fully and one partially occupied metal binding site per protomer according to a recent crystal structure. 4 AmpT and closely related peptidases (also known as MEROPS family M29 peptidases) do not resemble "standard" metallopeptidases with an H-E-x-x-H or H-x-x-E-H motif in sequence or structure, and have therefore recently been grouped together in the new MEROPS peptidase clan MQ.…”

Section: Introductionmentioning

confidence: 99%

Substrate Access to the Active Sites in Aminopeptidase T, a Representative of a New Metallopeptidase Clan

Odintsov

Sabala

Bourenkov

et al. 2005

Journal of Molecular Biology

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Isolation and characterization of a thermostable aminopeptidase(aminopeptidase T) from Thermus aquaticus YT-1, an extremely thermophilic bacterium.

Cited by 21 publications

References 5 publications

The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

A thermo‐stable lysine aminopeptidase from Pseudomonas aeruginosa: Isolation, purification, characterization, and sequence analysis

Substrate Access to the Active Sites in Aminopeptidase T, a Representative of a New Metallopeptidase Clan

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