Combinatorial manipulation of three key active site residues in glycinamide ribonucleotide transformylase

The enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2) haspreviously been shown to have three key polar active site residuesimportant for catalysis: N106, H108 and D144. Mutations of any of thesethree residues lead to substantially decreased catalytic activity, althoughnone of them are completely irreplaceable. In order to determine whetherany alternative arrangement of amino acids at these three positions couldlead to an active protein, all three of these residues were simultaneouslysubjected to saturation site-directed mutagenesis. The resultingcombinatorial library of mutant genes was screened for those encodingactive proteins using functional complementation. Glycinamideribonucleotide transformylase was found to be capable of tolerating no morethan one mutation amongst these key residues, since the only proteins foundto be sufficiently active to allow growth of auxotrophic cells on selectivemedia were the wild-type and enzymes containing a single mutation to one ofthese residues. It seems likely that no enzymes containing two or moremutations of these three residues possess significant catalytic activity.The combinatorial approach used could prove to be quite useful in proteinengineering and protein evolution experiments.