Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1.
2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.
Saku
Takashi
T
Department ofBiotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Fushinobu
Shinya
S
Jun
So-Young
SY
Ikeda
Naohiro
N
Nojiri
Hideaki
H
Yamane
Hisakazu
H
Omori
Toshio
T
Wakagi
Takayoshi
T