title: Combinatorial manipulation of three key active site residues in glycinamide ribonucleotide transformylase.
abstract: The enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2) has previously been shown to have three key polar active site residues important for catalysis: N106, H108 and D144. Mutations of any of these three residues lead to substantially decreased catalytic activity, although none of them are completely irreplaceable. In order to determine whether any alternative arrangement of amino acids at these three positions could lead to an active protein, all three of these residues were simultaneously subjected to saturation site-directed mutagenesis. The resulting combinatorial library of mutant genes was screened for those encoding active proteins using functional complementation. Glycinamide ribonucleotide transformylase was found to be capable of tolerating no more than one mutation amongst these key residues, since the only proteins found to be sufficiently active to allow growth of auxotrophic cells on selective media were the wild-type and enzymes containing a single mutation to one of these residues. It seems likely that no enzymes containing two or more mutations of these three residues possess significant catalytic activity. The combinatorial approach used could prove to be quite useful in protein engineering and protein evolution experiments.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Warren
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology; D015723:Gene Library; D019877:Hydroxymethyl and Formyl Transferases; D016297:Mutagenesis, Site-Directed; D051026:Phosphoribosylglycinamide Formyltransferase; D015202:Protein Engineering; D013329:Structure-Activity Relationship
chemical_list: D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases