Abstract
The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.
Highlights
Metallo--lactamases are bacterial enzymes that hydrolyze antibiotics of the -lactam family
The two subclass B3 -lactamases exhibit a broad activity spectrum against -lactam antibiotics, but FEZ-1 shows a preference for cephalosporins [18], whereas the L1 -lactamase seems to be more active against penicillins [19, 20]
We propose that the replacement of the tyrosine side chain by a methyl group, which increases the space in the vicinity of the active site, changes the position of the antibiotic in the catalytic pocket
Summary
Metallo--lactamases are bacterial enzymes that hydrolyze antibiotics of the -lactam family. In L1, the -substituent on C-6 or C-7 of the -lactam substrate generally fits in a hydrophobic pocket formed by the “flap” connecting 3 and 4, and by the loop between 3 and 8, which is considerably longer in subclass B3 enzymes than in subclass B1 [21, 22] In this pocket, the hydrophobic residues Phe156 and Ile162 of L1 are replaced by a Tyr and a Ser residue, respectively, in FEZ-1. CCTATGCTCATTTTGATGCTGCGGCCGGTAGCG CGCTACCGGCCGCAGCATCAAAATGAGCATAGG ATCAGGCCGTAATTATAGGAGCTATTGGCGTAAATCCTTGGG CCCAAGGATTTACGCCAATAGCTCCTATAATTACGGCCTGAT CCTGGACACACTAGAGGCGCTACCACCTGGACAATGAAAC GTTTCATTGTCCAGGTGGTAGCGCCTCTAGTGTGTCCAGG CTGTCTGGCGGTAAATCTGATTTTCATGCTGCTAATGATTCC GGAATCATTAGCAGCATGAAAATCAGATTTACCGCCAGACAG CATTATGCTAATGATTCCGCTACTTATTTTACTCAGAGTACTGTGGAT ATCCACAGTACTCTGAGTAAAATAAGTAGCGGAATCATTAGCATAATG GGAAGTATTGGCGTAGCTCCTGGGTATAAATTGGTT AACCAATTTATACCCAGGAGCTACGCCAATACTTCC GGAGGTATTGGCGTAAATCCTGGGGCTAAATTGGTTGATAAT ATTATCAACCAATTTAGCCCCAGGATTTACGCCAATACCTCC fide bridge between Cys256 and Cys296 These residues are conserved in FEZ-1, but this enzyme contains an additional Cys200 close to the active site. We have studied the role of residues His121, Tyr156, Ser221, Asn225, and Tyr228 in the catalytic activity of FEZ-1
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