Assembly of an active enzyme by the linkage of two protein modules.
The feasibility of creating new enzyme activities from enzymes of known function has precedence in view of protein evolution based on the concepts of molecular recruitment and exon shuffling. The enzymes encoded by the Escherichia coli genes purU and purN, N10-formyltetrahydrofolate hydrolase and glycinamide ribonucleotide (GAR) transformylase, respectively, catalyze similiar yet distinct reactions. N10-formyltetrahydrofolate hydrolase uses water to cleave N10-formyltetrahydrofolate into tetrahydrofolate and formate, whereas GAR transformylase catalyses the transfer of formyl from N10-formyltetrahydrofolate to GAR to yield formyl-GAR and tetrahydrofolate. The two enzymes show significant homology (approximately 60%) in the carboxyl-terminal region which, from the GAR transformylase crystal structure and labeling studies, is known to be the site of N10-formyltetrahydrofolate binding. Hybrid proteins were created by joining varying length segments of the N-terminal region of the PurN gene (GAR binding region) and the C-terminal (N10-formyltetrahydrofolate binding) region of PurU. Active PurN/PurU hybrids were then selected for by their ability to complement an auxotrophic E. coli strain. Hybrids able to complement the auxotrophs were purified to homogeneity and assayed for activity. The specific activity of two hybrid proteins was within 100- to 1000-fold of the native purN GAR transformylase validating the approach of constructing an enzyme active site from functional parts of others.
Nixon
A E
AE
Department of Chemistry, Pennsylvania State University, University Park 16802-6300, USA.
Warren
M S
MS
Benkovic
S J
SJ
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D019143:Evolution, Molecular; D005816:Genetic Complementation Test; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D051026:Phosphoribosylglycinamide Formyltransferase; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases; D000581:Amidohydrolases; C484016:purU protein, E coli