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Title 

On the structural and functional modularity of glycinamide ribonucleotide formyltransferases

Authors 

Seung Goo LeeS LutzS J Benkovic

Publisher 

Cold Spring Harbor Laboratory Press

Issue Date 

2003

Citation 

Protein Science, vol. 12, no. 10, pp. 2206-2214

Keywords 

domain swappingITCHYmodularityprotein engineeringSCRATCHYphosphoribosylglycinamide formyltransferasegene expressiongene fusiongene overexpressionhybridization

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 KM 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.

ISSN 

0961-8368

Link 

http://dx.doi.org/10.1110/ps.03139603

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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