1GHD

Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing

Summary for 1GHD

• Entry DOI: 10.2210/pdb1ghd/pdb
• Descriptor: GLUTARYL-7-AMINOCEPHALOSPORANIC ACID ACYLASE (3 entities in total)
• Functional Keywords: cephalosporin acylase, hydrolase
• Biological source: Pseudomonas sp. 130; More
• Total number of polymer chains: 2
• Total formula weight: 77593.11

Authors

Ding, Y.,Jiang, W.,Mao, X.,He, H.,Zhang, S.,Tang, H.,Bartlam, M.,Ye, S.,Jiang, F.,Liu, Y.,Zhao, G.,Rao, Z. (deposition date: 2000-12-07, release date: 2003-07-08, Last modification date: 2024-11-06)

Primary citation

Huang, X.,Zeng, R.,Ding, X.,Mao, X.,Ding, Y.,Rao, Z.,Xie, Y.,Jiang, W.,Zhao, G.
Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130.
J.Biol.Chem., 277:10256-10264, 2002

PubMed Abstract: Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis.

PubMed: 11782466
DOI: 10.1074/jbc.M108683200

Experimental method

X-RAY DIFFRACTION (2.4 Å)