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

Andrew E. Nixon, Stephen J. Benkovic

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

A hybrid glycinamide ribonucleotide transformylase was assembled from two protein 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, formyl 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 approach was used to improve the solubility and formyl transfer properties of the hybrid enzyme. The mutagenized hybrid glycinamide ribonucleotide transformylase was initially expressed as a fusion to the α-peptide of β-galactosidase. Clones were selected for improvement in solubility by determining which clones were capable of α-complementation using a blue/white screen. One clone was further characterized and found to have an improved efficiency of transfer of the ribonucleotide increasing this property to > 95%.

Original languageEnglish (US)
Pages (from-to)323-327
Number of pages5
JournalProtein Engineering
Volume13
Issue number5
DOIs
StatePublished - 2000

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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