Affinity labeled glutaryl-7-amino cephalosporanic acid acylase C130 can hydrolyze the inhibitor during crystallization.
7Beta-bromoacetyl amino cephalosporanic acid (BA-7-ACA), an analog of glutaryl-7-amino cephalosporanic acid (GL-7-ACA), can inhibit and specifically alkylate GL-7-ACA acylase (C130) from Pseudomonas sp.130, forming a carbon-carbon bond between BA-7-ACA and the C-2 on indole ring of Trp-beta4 residue of C130. Here we reported that BA-7-ACA labeled C130 (BA-C130) could self-catalyze the hydrolysis of BA-7-ACA during crystallization process. The hydrolysis was confirmed to be a reaction analogous to the one of GL-7-ACA by comparative matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry analysis. BA-C130 was inactive at room temperature, but in the process of crystallization at 18 degrees C it catalyzed the hydrolysis of BA-7-ACA, and thus made the latter become a substrate. Meanwhile, in crystals, 7-ACA was released but the acetic acid still bound with Trp-beta4, and as a result, the enzyme remained to be inactive. These results demonstrated that Trp-beta4 in the alphabetabetaalpha motif was critical and sensitive for the activity of C130 and also suggested that there was a conformational change induced by deacylation during the process of crystallization.
Zhang
Wei
W
Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, PR China.
Huang
Xi
X
Zhao
Guoping
G
Jiang
Weihong
W
D019342:Acetic Acid / Q000737:chemistry; D000478:Alkylation; D020816:Amino Acid Motifs; D000645:Ammonium Sulfate / Q000737:chemistry; D002384:Catalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000737:chemistry; D008956:Models, Chemical; D010399:Penicillin Amidase / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D011549:Pseudomonas / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase; D019342:Acetic Acid; D000645:Ammonium Sulfate