Affinity labeled glutaryl-7-amino cephalosporanic acid acylase C130 can hydrolyze the inhibitor during crystallization |
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Authors: | Zhang Wei Huang Xi Zhao Guoping Jiang Weihong |
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Institution: | 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. |
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Abstract: | 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. |
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Keywords: | BA-7-ACA GL-7-ACA BA-C130 C130 Crystallization |
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