PEDS Advance Access published online on November 22, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi071
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1 Laboratory Services Division, University of Guelph, Guelph, Ontario, Canada NIH 8J7
* To whom correspondence should be addressed. The hippurate hydrolase enzyme of Campylobacter jejuni was expressed in Escherichia coli as a six-histidine-tagged fusion protein. The purified recombinant enzyme was characterized to gain an understanding of the structure and activity of the hippurate hydrolase. The recombinant enzyme had a native molecular mass of 193 ± 11 kDa a reduced molecular mass of 42.4 ± 0.8 kDa, and possessed 1.98 ± 0.68 molecules of zinc per enzyme subunit molecule, suggesting that it was a homotetramer with two associated zinc ions. The enzyme was a metallocarboxypeptidase that was sensitive to silver, copper and ferrous ions, and displayed optimal activity at pH 7.5 and 50°C. It hydrolyzed carboxypeptidase substrates in vitro, displaying its highest activity against N-benzoyl-linked small aliphatic amino acids. A high proportion of the enzyme structure consisted of highly ordered
Received May 11, 2005
Revised August 10, 2005
Accepted September 12, 2005
Article
Enzymatic activity of Campylobacter jejuni hippurate hydrolase
M. Steele 1,
M. Marcone 2,
C. Gyles 3,
V. L. Chan 4,
and
J. Odumeru 1 *
2 Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
3 Pathobiology Department, University of Guelph, Guelph, Ontario, Canada N1G 2W1
4 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
J. Odumeru, E-mail: jodumeru{at}lsd.uoguelph.ca
Abstract 
-helix and 
-sheet sequences. An alignment of the amino acid sequence of the hippurate hydrolase enzyme with those of related enzymes with similar activities revealed several conserved amino acids, which might be involved in enzyme catalysis or metal ion binding for the enzyme. Site-directed mutagenesis of the recombinant enzyme demonstrated that the Asp76, Aps104, Glu134, Glu135, His161 and His356 positions were important for the catalytic activity of the enzyme.
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