title: Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue.
abstract: We describe a procedure by which the codon (AGC) for the active-site serine-70 of pBR322 beta-lactamase (penicillinase, penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is altered to that for cysteine (TGC). The pertinent nucleotide bases, A-G-C-A, positions 410-413, of pBR322 are excised by treating a limited HgiAI digest of pBR322 with the 3' leads to 5' exonuclease of T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in two steps. First, the Kpn I molecular linker d(T-G-G-T-A-C-C-A) is ligated into the gap described above. The internal sequence G-T-A-C is then excised enzymatically with Kpn I and T4 DNA polymerase and the molecule is recircularized. This mutant gene, which codes for a thiol-beta-lactamase, confers on Escherichia coli K-12 hosts an ampicillin resistance that is reduced compared with that given by pBR322 yet is greater than that of E. coli lacking any intact beta-lactamase gene. Cell-free extracts of E. coli strains hosting the thiol-beta-lactamase gene possess a p-chloromercuribenzoate-sensitive beta-lactamase activity.
mesh_terms: D000595:Amino Acid Sequence; D000667:Ampicillin / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology; D005796:Genes; D005818:Genetic Engineering; D010403:Penicillin Resistance; D012694:Serine; D013329:Structure-Activity Relationship; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D002729:Chloromercuribenzoates; D065093:beta-Lactamase Inhibitors; D012694:Serine; D000667:Ampicillin; D001618:beta-Lactamases; D003545:Cysteine