Site-directed mutagenesis techniques in the study of Escherichia coli serine hydroxymethyltransferase.
The 3340-bp fragment containing the Escherichia coli glyA gene coding for serine hydroxymethyltransferase was reduced in size by PCR, and the 1600-bp fragment obtained was cloned into the vector pBR322 in both orientations (5'-3', and 3'-5'). This DNA manipulation allowed us to perform site-directed mutagenesis by PCR on the glyA gene. To overcome the problem of the presence of wild-type protein in the various mutant enzyme preparations, the E. coli strain GS245 used to express recombinant serine hydroxymethyltransferase was made recA deficient through generalized transduction mediated by phage P1. The new strain was used for the production of a mutant form of the enzyme, in which the pyridoxal 5'-phosphate binding lysine was substituted by a glutamine. The preparation of this mutant form was completely devoid of wild-type enzyme contamination and measurements of its catalytic activity in the transamination reactions of L- and D-alanine confirmed the suggestion that the active site lysine is not the base that removes the alpha-proton from the substrate.
Iurescia
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Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Roma, Italy.
Condò
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Angelaccio
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Delle Fratte
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Bossa
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D000409:Alanine / Q000494:pharmacology; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005973:Glutamine / Q000737:chemistry; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics
D011994:Recombinant Proteins; D005973:Glutamine; D012696:Glycine Hydroxymethyltransferase; D000409:Alanine