The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

Yifat Fundoiano-Hershcovitz; Larisa Rabinovitch; Smadar Shulami; Vera Reiland; Gil Shoham; Yuval Shoham

doi:10.1016/j.femsle.2004.12.001

The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

Yifat Fundoiano-Hershcovitz, Larisa Rabinovitch, Smadar Shulami, Vera Reiland, Gil Shoham, Yuval Shoham^*

^*Corresponding author for this work

Institute of Chemistry

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80°C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl₂ was able to restore activity, and the binding stochiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in k _cat/K_m values of 1-5 × 10¹ s^-1 mM^-1.

Original language	English
Pages (from-to)	157-163
Number of pages	7
Journal	FEMS Microbiology Letters
Volume	243
Issue number	1
DOIs	https://doi.org/10.1016/j.femsle.2004.12.001
State	Published - 1 Feb 2005

Bibliographical note

Funding Information:
This study was supported by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany) and the Israel Science Foundation (ISF). V.R. was supported by a Marie Curie Fellowship of the European Commission and by a Lady Davis Fellowship from the Hebrew University of Jerusalem, Israel.

Keywords

Bacillus subtilis aminopeptidase
Catalytic mechanism
Substrate specificity
Zn-metalloenzyme
ywad gene

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10.1016/j.femsle.2004.12.001

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abstract = "The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80°C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stochiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in k cat/Km values of 1-5 × 101 s-1 mM-1.",

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AU - Reiland, Vera

AU - Shoham, Gil

AU - Shoham, Yuval

N1 - Funding Information: This study was supported by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany) and the Israel Science Foundation (ISF). V.R. was supported by a Marie Curie Fellowship of the European Commission and by a Lady Davis Fellowship from the Hebrew University of Jerusalem, Israel.

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The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

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Keywords

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