A triple mutant in the Ω-loop of TEM-1 β-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis

Abstract

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism.

Keywords: Antibiotic Resistance; Beta-Lactamase; Enzyme Catalysis; Enzyme Evolution; Enzyme Kinetics; Enzyme Structure; Protein Stability; Protein Structure; X-ray Crystallography.

FIGURE 1.

Progress curves of ceftazidime hydrolysis by the TEM-1 W165Y/E166Y/P167G enzyme. The experiments were performed in triplicate. The error bars are omitted for clarity (S.D. less than 5%). The line represents the fit of progress curve to the branched pathway equation for ceftazidime hydrolysis (diamonds) with the inclusion of 60% of saturated (NH₄)₂SO₄ (circles). The increase of the burst in the presence of saturated (NH₄)₂SO₄ is indicative of the branched pathway.

FIGURE 2.

pH dependence of k_cat/K_m (A), k_cat (B), and K_m (C) for wild-type TEM-1 (filled circles, left y axis) and the W165Y/E166Y/P167G triple mutant (empty circles, right y axis) β-lactamases for hydrolysis of nitrocefin. The triple mutant exhibits a shift of two pH units in its optimal pH for k_cat/K_m and nearly two pH units for k_cat compared with wild-type TEM-1 β-lactamase (A and B). The error bars in the plots represent standard deviations for each data point.

FIGURE 3.

Crystal structure of the TEM-1 W165Y/E166Y/P167G/L201P mutant. A, top panel, TEM-1 in dark cyan (PDB code 1XPB, 1.9 Å resolution) aligned with the TEM-1 W165Y/E166Y/P167G/L201P (salmon) enzyme. The active-site Ser-70 is represented in both structures in stick model. The simulated annealing omit difference map contoured at ∼3 σ for residues 164–174 in the W165Y/E166Y/P167G/L201P structure is shown as a gray mesh and reveals the two different conformations that the Tyr-166 adopts in the W165Y/E166Y/P167G/L201P mutant structure. Bottom panels, detailed view of the catalytic apparatus of TEM-1 (left bottom panel) and proposed apparatus of W165Y/E166Y/P167G/L201P mutant (right bottom panel). In the mutant, the catalytic water molecule is not present because of the bulkier tyrosine residue and the movement of Asn-170. B, top panel, 120° rotation of the structure alignment with the dotted black box indicating the Ω-loop. Bottom panels, view of the Ω-loop electrostatic network. The Ω-loop structure of the mutant is maintained by the preserved salt bridges among the charged residues within the Ω-loop. C, surface representation of TEM-1 (dark cyan) (PDB code 1XPB) and W165Y/E166Y/P167G/L201P (salmon) structures. The active site Ser-70 and Asn-170 are labeled and represented in fluorescent green. The active site cavity of the mutant is enlarged and elongated, forming an L shape. In contrast, the TEM-1 active site is shallow and narrow.

FIGURE 4.

Temperature factors of the main chain of W165Y/E166Y/P167G/L201P (salmon) and TEM-1 (PDB code 1XPB) (dark cyan). The corresponding β-lactamase structures are shown above the temperature factor curves. On the left in dark cyan is TEM-1, and on the right is W165Y/E166Y/P167G/L201P in salmon. The Ω-loop is outlined in the black boxes. The peak in the temperature factor curve corresponds to an increase in the B-factors of residues 167–172 in W165Y/E166Y/P167G/L201P. The numbering follows the conventional numbering for class A β-lactamases.

FIGURE 5.

Alignment of the three structures of the TEM-1 W165Y/E166Y/P167G mutants solved in this study. In salmon is the structure of W165Y/E166Y/P167G/L201P. The structure of W165Y/E166Y/P167G/M182T is shown in gold. Residues 168–174 of the Ω-loop of W165Y/E166Y/P167G/M182T showed very little density, and were not modeled in the final structure (residues 167 and 175 are connected with a dashed line). The structure of S70G/W165Y/E166Y/P167G is shown in light blue. The Tyr-166 residue assumes the same conformation in the three structures; however, in the L201P structure, an addition conformation of Tyr-166 is observed. The inset shows a detailed view of the Ω-loop in the three structures, and the simulated annealing omit difference maps contoured at ∼3 σ are shown as a gray mesh for residues 164–174 in the W165Y/E166Y/P167G/L201P and S70G/W165Y/E166Y/P167G structures and for residues 164–167 in the W165Y/E166Y/P167G/M182T structure.

FIGURE 6.

Docking results of ceftazidime with wild-type TEM-1 (A, dark cyan) and the W165Y/E166Y/P167G/L201P mutant (B, salmon). Ser-70 and Asn-170 are shown in fluorescent green in both structures. The same constraints were used for the docking parameters in Autodock Vina for both structures with an exhaustiveness of eight. The conformations with the lowest binding energy are shown. The movement of the Ω-loop in the triple mutant expands the active site, forming an L-shaped cavity (bottom panels) that provides more space to accommodate ceftazidime than in wild-type TEM-1.

FIGURE 7.

Ω-Loop structures in TEM-1, TEM-64, and TEM W165Y/E166Y/P167G/L201P. The structural conformation of the Ω-loop in TEM-1 (PDB code 1XPB) is shown in dark cyan, TEM-64 (PDB code 1JWZ) is in gray, and TEM W165Y/E166Y/P167G/L201P is in salmon. Represented in stick model are Glu-166 in TEM-1 and TEM-64, Tyr-166 in TEM W165Y/E166Y/P167G/L201P, and Asn-170 in all three enzymes. The position of the side chain at position 166 is very similar in all structures, but Asn-170 shows a great degree of variability. The three enzymes exhibit significant differences with respect to Ω-loop position and conformation. The TEM-1 W165Y/E166Y/P167G/L201P mutant enzyme has an Ω-loop conformation that results in wider active site pocket compared with both TEM-1 and TEM-64.