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PEDS Advance Access originally published online on November 22, 2005
Protein Engineering Design and Selection 2006 19(1):17-25; doi:10.1093/protein/gzi071
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Enzymatic activity of Campylobacter jejuni hippurate hydrolase

M. Steele1, M. Marcone2, C. Gyles3, V.L. Chan4,5 and J. Odumeru1,6

1Laboratory Services Division, University of Guelph, Guelph, Ontario, Canada NIH 8J7, 2Department of Food Science and 3Pathobiology Department, University of Guelph, Guelph, Ontario, Canada N1G 2W1 and Departments of 4Medical Genetics and Microbiology and 5Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8

6 To whom correspondence should be addressed. E-mail: jodumeru{at}lsd.uoguelph.ca

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 {alpha}-helix and ß-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.

Keywords: amidohydrolase/carboxypeptidase/hippurate hydrolase/mutagenesis

Received May 11, 2005; revised August 10, 2005; accepted September 12, 2005.

Edited by Alan Berry


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