Improvement in the efficiency of formyl transfer of a GAR transformylase hybrid enzyme

A hybrid glycinamide ribonucleotide transformylase was assembled from two p rotein domains that were treated as discrete modules. One module contained the ribonucleotide binding domain from the purN glycinamide ribonucleotide transformylase; the second module contained the catalytic machinery and the formyl tetrahydrofolate binding domain from the enzyme encoded by purU, fo rmyl tetrahydrofolate hydrolase. The resultant enzyme showed 0.1% catalytic activity of the wild-type glycinamide ribonucleotide transformylase enzyme but had a formyl transfer efficiency of 10%. A combinatorial mutagenesis a pproach was used to improve the solubility and formyl transfer properties o f the hybrid enzyme. The mutagenized hybrid glycinamide ribonucleotide tran sformylase was initially expressed as a fusion to the alpha-peptide of beta -galactosidase. Clones were selected for improvement in solubility by deter mining which clones were capable of alpha-complementation using a blue/whit e screen. One clone was further characterized and found to have an improved efficiency of transfer of the ribonucleotide increasing this property to > 95%.