Enzymatic activity of Campylobacter jejuni hippurate hydrolase.
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 degrees 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 beta-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 Asp(76), Aps(104), Glu(134), Glu(135), His(161) and His(356) positions were important for the catalytic activity of the enzyme.
Steele
M
M
Laboratory Services Division, University of Guelph, Guelph, Ontario, Canada NIH 8J7, Department of Food Science, Guelph, Ontario, Canada N1G 2W1.
Marcone
M
M
Gyles
C
C
Chan
V L
VL
Odumeru
J
J
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D016123:Campylobacter jejuni / Q000201:enzymology*; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002412:Cations; D003300:Copper / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D012834:Silver / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D000596:Amino Acids; D002412:Cations; D012834:Silver; D006639:Histidine; D003300:Copper; D007501:Iron; D002268:Carboxypeptidases; D000581:Amidohydrolases; C021356:hippurate hydrolase; D015032:Zinc