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

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 alpha-peptide of beta-galactosidase. Clones were selected for improvement in solubility by determining which clones were capable of alpha-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%.

[1]  R. Huber,et al.  New enzyme lineages by subdomain shuffling. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[2]  H. K. Schachman,et al.  Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: Implications for domain switching , 1996, Protein science : a publication of the Protein Society.

[3]  J. H. Shim,et al.  Combinatorial protein engineering by incremental truncation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. H. Shim,et al.  Evaluation of the kinetic mechanism of Escherichia coli glycinamide ribonucleotide transformylase. , 1998, Biochemistry.

[5]  S. Benkovic,et al.  Assembly of an active enzyme by the linkage of two protein modules. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Manuel C. Peitsch,et al.  Protein Modeling by E-mail , 1995, Bio/Technology.

[7]  S. Benkovic,et al.  A rapid screen of active site mutants in glycinamide ribonucleotide transformylase. , 1996, Biochemistry.

[8]  W. Gilbert,et al.  The exon theory of genes. , 1987, Cold Spring Harbor symposia on quantitative biology.

[9]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[10]  P. Nygaard,et al.  Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli , 1993, Journal of bacteriology.

[11]  W. Gilbert.  Why genes in pieces? , 1978, Nature.

[12]  M. Ashburner.  A Laboratory manual , 1989 .

[13]  I. Wilson,et al.  Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 A resolution. , 1995, Journal of molecular biology.

[14]  W. Stemmer,et al.  Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling , 1996, Nature Biotechnology.

[15]  S. Benkovic,et al.  Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase. , 1990, Biochemistry.

[16]  R. Jensen.  Enzyme recruitment in evolution of new function. , 1976, Annual review of microbiology.

[17]  S. Benkovic,et al.  Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli , 1995, Journal of bacteriology.

[18]  S. Benkovic,et al.  Incremental truncation as a strategy in the engineering of novel biocatalysts. , 1999, Bioorganic & medicinal chemistry.

[19]  G. Petsko,et al.  On the origin of enzymatic species. , 1993, Trends in biochemical sciences.