The diversity in substrate recognition spectra exhibited by various beta-lactamases can result from one or a few mutations in the active-site area. Using @entity526 @entity49390 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 @entity5898 , @entity3524 ( @entity3525 ), 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 @entity3525 and decreased @entity114 resistance. Although selection was undertaken with @entity3525 , productive binding (K(M)) was improved for all substrates tested, including @entity3397 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 @entity48627 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 @entity48627 but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the @entity18585 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.

High tolerance to simultaneous active-site mutations in XXXX : Distinct mutational paths provide more generalized beta-lactam recognition.

@entity3525 :: ('CHEBI:36467', 'Chemical') :: ['CTX'] @entity18585 :: ('2716540(Tax:562)', 'Gene') :: ['TEM-1'] @entity114 :: ('MESH:D010406', 'Chemical') :: ['penicillin'] @entity5898 :: ('CHEBI:23066', 'Chemical') :: ['cephalosporin'] @entity49390 :: ('13905334', 'Gene') :: ['TEM-1 beta-lactamase'] @entity3524 :: ('MESH:D002439', 'Chemical') :: ['cefotaxime'] @entity3397 :: ('MESH:D010400', 'Chemical') :: ['benzylpenicillin'] @entity48627 :: ('p|SUB|G|238|S', 'ProteinMutation') :: ['G238S'] @entity526 :: ('562', 'Species') :: ['Escherichia coli']
@entity49390 :: (13905334,Gene) :: ['TEM-1 beta-lactamase']
