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Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C

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Fritz-Wolf,  Karin
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Kabsch,  Wolfgang
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Fritz-Wolf, K., Koller, K.-P., Lange, G., Liesum, A., Sauber, K., Schreuder, H., et al. (2002). Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C. Protein Science, 11(1), 92-103. doi:10.1110/ps.27502.


Cite as: https://hdl.handle.net/11858/00-001M-0000-0029-3132-6
Abstract
Glutarylamidase is an important enzyme employed in the commercial production of 7-aminocephalosporanic acid, a starting compound in the synthesis of cephalosporin antibiotics. 7-aminocephalosporanic acid is obtained from cephalosporin C, a natural antibiotic, either chemically or by a two-step enzymatic process utilizing the enzymes D-amino acid oxidase and glutarylamidase. We have investigated possibilities for redesigning glutarylamidase for the production of 7-aminocephalosporanic acid from cephalosporin C in a single enzymatic step. These studies are based on the structures of glutarylamidase, which we have solved with bound phosphate and ethylene glycol to 2.5 Å resolution and with bound glycerol to 2.4 Å. The phosphate binds near the catalytic serine in a way that mimics the hemiacetal that develops during catalysis, while the glycerol occupies the side-chain binding pocket. Our structures show that the enzyme is not only structurally similar to penicillin G acylase but also employs essentially the same mechanism in which the α-amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the Oγ through a hydrogen bond relay network involving the α-amino group of the serine and a bound water molecule. A plausible model of the enzyme–substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7-aminocephalosporanic acid.