PMID:14500878
| Citation |
Lee, SG, Lutz, S and Benkovic, SJ (2003) On the structural and functional modularity of glycinamide ribonucleotide formyltransferases. Protein Sci. 12:2206-14 |
|---|---|
| Abstract |
Glycinamide ribonucleotide formyltransferases (GARTs) are part of the de novo purine biosynthetic pathway, catalyzing the direct transfer of a formyl group from the tetrahydrofolate cofactor to the glycinamide ribonucleotide substrate. Despite the low amino acid-sequence identity between the GARTs from Escherichia coli and human, their tertiary structures are superimposable. As part of our functional studies of these enzymes, we have investigated the interchangeability of individual protein fragments or modules between the two enzymes and the functional properties of the resulting hybrids. The modular nature of GART facilitated the creation of combinatorial libraries of chimeras between the Escherichia coli and human enzymes, which were functionally selected through complementation of an auxotrophic Escherichia coli strain. From a pool of several dozen sequence distinct hybrids, six in vivo-functional fusion genes were selected, overexpressed, and purified to homogeneity. The kinetic analysis of these constructs and the comparison of their k(cat) and K(M) values to the parental enzymes suggest that the characteristic kinetic properties from the two parents are "modular encoded" and can be exchanged by domain swapping. The chimeras in general, however, are subject to temperature instability and misfolding; thus, they serve primarily as useful candidates for further rounds of optimization. |
| Links |
PubMed PMC2366928 Online version:10.1110/ps.03139603 |
| Keywords |
Catalysis; Catalytic Domain/genetics; DNA Shuffling; Escherichia coli/genetics; Escherichia coli/metabolism; Folic Acid/analogs & derivatives; Folic Acid/metabolism; Gene Expression Regulation, Enzymologic; Humans; Hydrolysis; Hydroxymethyl and Formyl Transferases/chemistry; Hydroxymethyl and Formyl Transferases/genetics; Hydroxymethyl and Formyl Transferases/metabolism; Kinetics; Models, Molecular; Phosphoribosylglycinamide Formyltransferase; Protein Binding; Protein Engineering; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism; Structural Homology, Protein; Structure-Activity Relationship; Temperature |
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