His23beta and Glu455beta of the Pseudomonas sp. 130 glutaryl-7-amino cephalosporanic acid acylase are crucially important for efficient autoproteolysis and enzymatic catalysis.
Glutaryl-7-amino cephalosporanic acid acylase is a member of the N-terminal nucleophilic hydrolase family of enzymes. The crystal structure of the acylase reveals there is a Ser-His-Glu motif composed of Ser1beta, His23beta, and Glu455beta near the active site. This mimics the catalytic triad of Ser-His-Asp in serine proteases. Experiments prove that maturation of this enzyme involves autoproteolysis. It has been shown that Ser1beta is the catalytic residue for the autoproteolysis and catalytic reaction. Our works on site-directed mutagenesis followed by the characterization of mutant enzymes demonstrated that His23beta is essential for autoproteolysis whereas Glu455beta is responsible for the efficiency of the process. Neither His23beta nor Glu455beta is essential for the acylase activity, although they affect the catalytic efficiency.
Mao
Xiang
X
Laboratory of Microbial Molecular Physiology, Shanghai Institutes for Biological Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. xmao@mdanderson.org
Wang
Weiwu
W
Jiang
Weihong
W
Zhao
Guo-Ping
GP
D000595:Amino Acid Sequence; D002511:Cephalosporins / Q000378:metabolism; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D011549:Pseudomonas / Q000201:enzymology*
D002511:Cephalosporins; C081063:glutaryl-7-aminocephalosporanic acid; D018698:Glutamic Acid; D006639:Histidine; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase