High tolerance to simultaneous active‐site mutations in TEM‐1 β‐lactamase: Distinct mutational paths provide more generalized β‐lactam recognition

Abstract

The diversity in substrate recognition spectra exhibited by various b-lactamases can result from one or a few mutations in the active-site area. Using Escherichia coli TEM-1 b-lactamase as a template that efficiently hydrolyses penicillins, we performed site-saturation mutagenesis simultaneously on two opposite faces of the active-site cavity. Residues 104 and 105 as well as 238, 240, and 244 were targeted to verify their combinatorial effects on substrate specificity and enzyme activity and to probe for cooperativity between these residues. Selection for hydrolysis of an extended-spectrum cephalosporin, cefotaxime (CTX), led to the identification of a variety of novel mutational combinations. In vivo survival assays and in vitro characterization demonstrated a general tendency toward increased CTX and decreased penicillin resistance. Although selection was undertaken with CTX, productive binding (K M ) was improved for all substrates tested, including benzylpenicillin for which catalytic turnover (k cat ) was reduced. This indicates broadened substrate specificity, resulting in more generalized (or less specialized) variants. In most variants, the G238S mutation largely accounted for the observed properties, with additional mutations acting in an additive fashion to enhance these properties. However, the most efficient variant did not harbor the mutation G238S but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the TEM-1 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.

Combinatorial active‐site variants confer sustained clavulanate resistance in BlaC β‐lactamase from Mycobacterium tuberculosis

“…Enzyme kinetics assays were performed using the following wavelengths and molar extinction coefficients: BZ, 232 nm, 1000 M −1 cm −1 ; CF, 264 nm, 7960 M −1 cm −1 ; CRO, 260 nm, 9400 M −1 cm −1 ; FOX, 260 nm, 7700 M −1 cm −1 . All substrates used in the present study are presented in Figure .…”

Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

“…The purification involved an anion exchange step (HiPrep 16/10 QXL, GE Healthcare) followed by gel filtration (HiLoad 26/60 Superdex 75 prep grade, GE Healthcare). Proteins used for enzyme kinetics and circular dichroism (CD) characterization were also expressed and purified according to previously established protocols [30], [XPATH ERROR: unknown variable "next".]. Briefly, overexpression was performed in Luria-Bertani broth.…”

Structure‐based design of combinatorial mutagenesis libraries

“…As such, site saturation studies have helped to characterize functional and stability contributions of active site residues . Also, site‐saturation methods are widely popular for understanding diversity in the spectrum of substrate recognition …”