[Effect of site-directed mutagenesis on soluble expression and specific activity of amide hydrolase DamH].
OBJECTIVE
DamH is a bifunctional hydrolase that hydrolyzes the amide and ester bonds. Previous studies demonstrated that mutagenesis of non-catalytic residues shows a negative impact on the soluble expression and specific activity of DamH. Therefore, we studied the catalytic triad of DamH and the effect of non-catalytic mutagenesis on the soluble expression and specific activity of DamH.


METHODS
We performed site-directed mutation experiment of the 3 possible catalytic sites: S149, E244 and H274 and the non-active sites by using overlapping PCR. In the whole cell catalytic experiments, 2'-methyl-6'-ethyl-2-chloroacetanilide (CMEPA) hydrolase activities of the three mutants (S149A, E244A and H274A) were assayed. Mutants of D165 and N192 were purified by affinity chromatography of Ni²⁺-NTA. At the same time the hydrolase activities of mutants were compared with that of the wild-type strain.


RESULTS
S149-E244-H274 was the catalytic triad of DamH. Kinetics shows that the CMEPA hydrolase activity of mutant S149A declined to 5% of the wild-type strain and none of E244A and H274A mutants showed any CMEPA hydrolase activity. Mutations of D165 and N192 would affect the soluble expression of recombinant enzyme. The soluble expression levels of D165P and N192P were 28.2% and 20.8% of the wild-type strain, respectively. Furthermore, hydrolase activities of N192P and D165P were only 55.5% and 49.7% of the wild-type enzyme, respectively.


CONCLUSIONS
DamH uses the same active site to hydrolyze esters and amides.
Fei
F
Wang
Zhoukun
Z
Li
Jie
J
Zhou
Zhongli
Z
Cui
D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012995:Solubility
D000577:Amides; D029968:Escherichia coli Proteins; D006867:Hydrolases