1GK1

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

Summary for 1GK1

• Entry DOI: 10.2210/pdb1gk1/pdb
• Related: 1GK0
• Descriptor: CEPHALOSPORIN ACYLASE, GLYCEROL, ... (4 entities in total)
• Functional Keywords: hydrolase, cephalosporin acylase, glutaryl acylase, cephalosporin c, catalytic triad, ntn-hydrolase, x-raz structure
• Biological source: PSEUDOMONAS SP.; More
• Total number of polymer chains: 4
• Total formula weight: 150747.31

Authors

Fritz-Wolf, K.,Koller, K.P.,Lange, G.,Liesum, A.,Sauber, K.,Schreuder, H.,Aretz, W.,Kabsch, W. (deposition date: 2001-08-07, release date: 2002-01-01, Last modification date: 2023-12-13)

Primary citation

Fritz-Wolf, K.,Koller, K.P.,Lange, G.,Liesum, A.,Sauber, K.,Schreuder, H.,Aretz, W.,Kabsch, W.
Structure-Based Prediction of Modifications in Glutarylamidase to Allow Single-Step Enzymatic Production of 7-Aminocephalosporanic Acid from Cephalosporin C.
Protein Sci., 11:92-, 2002

PubMed 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 A resolution and with bound glycerol to 2.4 A. 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 alpha-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 O(gamma) through a hydrogen bond relay network involving the alpha-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.

PubMed: 11742126
DOI: 10.1110/PS.PS.27502

Experimental method

X-RAY DIFFRACTION (2.4 Å)