Probing the Specificity of the Subclass B3 FEZ-1 Metallo-β-lactamase by Site-directed Mutagenesis

Abstract

The subclass B3 FEZ-1 ␤-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the ␤-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. Metallo-␤-lactamases are bacterial enzymes that hydrolyze antibiotics of the ␤-lactam family. They are classified as class B (1) or group 3 (2) ␤-lactamases. In the last decade, the discovery of an increasing number of new metallo-␤-lactamases resulted in a subdivision into three molecular subclasses: B1, B2, and B3. Thereafter, a standard numbering scheme (3) was adopted that identifies conserved residues involved in the catalytic activity. Subclass B3 ␤-lactamases are broad spectrum enzymes that require one or two Zn (II) ions for activity (4). These enzymes are produced by various environmental species, of which some can cause opportunistic infections (such as S. maltophilia (5) and F. gormanii (6)), whereas others are not pathogenic such as Janthinobacterium lividum (7) and Caulobacter crescentus (8, 9). The structures of several subclass B1 ␤-lactamases have been solved by x-ray crystallography (BcII (10, 11), CcrA (12), BlaB (13), and IMP-1 (14)). To date, no structure of a subclass B2 enzyme is available. In subclass B3, the crystal structures of the metallo ␤-lactamases L1 from S. maltophilia (15) and FEZ-1 from F. gormanii (16) have been solved. Comparison of the tertiary structures of the different enzymes highlighted similar organizations of the secondary structure elements; they all contain an ␣␤␤␣ sandwich with two central ␤-sheets and ␣-helices on the external faces (10). The active site with the binuclear zinc center is located at the bottom of the ␤-sheet core. Zn1 is tetrahedrally coordinated by three histidines, His 116 , His 118 , His 196 , and a water molecule. In the subclass B1 enzymes, Zn2 is coordinated by His 263 , Asp 120 , Cys 221 , and two water molecules to form a trigonal bipyramid. In subclass B3 ␤-lactamases, a Ser residue replaces Cys 221 , and His 121 is the third ligand of Zn2. Our studies were performed on the FEZ-1 ␤-lactamase. FEZ-1 is a monomeric enzyme. The sequence of the mature protein is easily aligned with that of the L1 enzyme with 33% of isology (17). 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). Comparison of the x-ray structures reveals similar zinc binding sites in...

Structural Insights into the Subclass B3 Metallo-β-Lactamase SMB-1 and the Mode of Inhibition by the Common Metallo-β-Lactamase Inhibitor Mercaptoacetate

“…We hypothesized that various substrate preferences among the subclass B3 MBLs could be attributed to structural differences in the two loop regions. In fact, some of the amino acid residues in these two loop regions are already known to be responsible for binding to the substrate ␤-lactam antibiotics and/or inhibitor agents, as revealed by the complexed structures of BJP-1-4-nitrobenzenesulfonamide and L1-hydrolyzed moxalactam (34,42) and also by site-directed mutagenesis approaches (41,43).…”

In VivoImpact of Met221 Substitution in GOB Metallo-β-Lactamase

“…Position 221 is critical for the activities of all M␤Ls studied so far, which contain either a Cys ligand or a Ser residue (17,19,23,24,30,34). All GOB enzymes reported so far possess a Met residue at position 221.…”

Crystal Structure of the Mobile Metallo-β-Lactamase AIM-1 from Pseudomonas aeruginosa: Insights into Antibiotic Binding and the Role of Gln157

“…Furthermore, Asn233 is not uniformly conserved in B1 MBLs. In the B3 enzymes, Tyr228 (L1) and Asn225 (FEZ-1) have been variously proposed to stabilize oxyanionic species (22,56) but the effects of substitutions at these positions are also substrate dependent (9,40). Taken together, these data indicate that, although Zn1 is believed to polarize the ␤-lactam carbonyl for addition of W1 (44), involvement of other elements of the MBL active site in this process, or in stabilizing more transiently populated species, has not been demonstrated.…”