A point mutation leads to altered product specificity in beta-lactamase catalysis

Abstract

beta-Lactamases are the primary cause of beta-lactam antibiotic resistance in many pathogenic organisms. The beta-lactamase catalytic mechanism has been shown to involve a covalent acyl-enzyme. Examination of the structure of the class A beta-lactamase from Bacillus licheniformis suggested that replacement of Asn-170 by leucine would disrupt the deacylation reaction by displacing the hydrolytic water molecule. When N170L beta-lactamase was reacted with penicillins, a novel product was formed. We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine-oxazolinone as the initial product. The oxazolinone subsequently undergoes rapid breakdown leading to the formation of N-phenylacetylglycine and N-formylpenicillamine. This appears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered product, effectively changing the product specificity of beta-lactamase into that of D-Ala-D-Ala-carboxypeptidase interacting with benzylpenicillin.

Figure 1

Kinetics of wild-type (solid line) and N170L (dotted line) B. licheniformis β-lactamase-catalyzed degradation of benzylpenicillin (A) at 240 nm, (B) at 257 nm. (C) Spectra of benzylpenicillin (solid line), benzylpenicillin after hydrolysis with wild-type β-lactamase (dotted line), and benzylpenicillin after degradation with N170L β-lactamase (broken line): absorbance due to the enzyme has been subtracted.

Figure 2

Thiazolidine methyl region of the ¹H-NMR spectra of the N170L B. licheniformis β-lactamase catalyzed degradation of benzylpenicillin. (A) Just after completion of the first kinetic phase, and (B) 2.3 hr after completion of the first phase. Peak assignments: A, d-5,5-dimethyl-Δ²-thiazoline-4-carboxylic acid, Me; B, 5S,6R-benzylpenicilloic acid, Me; C, 5R,6R-benzylpenicilloic acid, Me; D, N-formylpenicillamine, Me; E, not identified, possibly benzylpenillic acid; F, d-5,5-dimethyl-Δ²-thiazoline-4-carboxylic acid and N-formyl-penicillamine, Me; G, 5R,6R-benzylpenicilloic acid, Me; H, 5S,6R-benzylpenicilloic acid, Me. Resonances from A and B not shown: δ 3.44 ppm, s, 5S,6R-benzylpenicilloic acid, H-9; δ 3.46 ppm, s, 5R,6R-benzylpenicilloic acid, H-9; δ 3.71 ppm, s, N-phenylacetylglycine, H-9; δ 3.74 ppm, dd, 12 Hz, 42 Hz, 5R,6R-benzylpenicilloic acid, H-9; δ 3.83 ppm, dd, 5 Hz, 26 Hz, 5S,6R-benzylpenicilloic acid, H-9; δ 4.27 ppm, d, 6 Hz, 5R,6R-benzylpenicilloic acid, H-6; δ 4.35 ppm, s, N-formylpenicillamine, H-3; δ 4.53 ppm, d, 2 Hz, d-5,5-dimethyl-Δ²-thiazoline-4-carboxylic acid, H-3; δ 5.09 ppm, d, 6 Hz, 5R,6R-benzylpenicilloic acid, H-5; δ 5.10 ppm, s, 5S,6R-benzylpenicilloic acid, H-5; δ 7.35–7.48 ppm, aromatics; δ 8.18 ppm, s, N-formylpenicillamine, H-5; δ 8.23 ppm, d, 2 Hz, d-5,5-dimethyl-Δ²-thiazoline-4-carboxylic acid, H-3.