About this item:

551 Views | 126 Downloads

Author Notes:

Correspondence: Ichiro Matsumura; Email: imatsum@emory.edu

Acknowledgments: We are greatly indebted to Dr. Baoyun Xia for his guidance in the HPLC analysis.

We also thank Dr. Monica Gerth for her assistance with the preparation of figures.

DNA sequencing was performed at the Center for Fundamental and Applied Molecular Evolution at Emory University.


Research Funding:

This work was supported by grants from the NIH (R01 GM074264), the NSF (CHE-0404677) and the Institute of Molecular Biosciences at Massey University.

A Study in Molecular Contingency: Glutamine Phosphoribosylpyrophosphate Amidotransferase is a Promiscuous and Evolvable Phosphoribosylanthranilate Isomerase


Journal Title:

Journal of Molecular Biology


Volume 377, Number 2


, Pages 323-336

Type of Work:

Article | Post-print: After Peer Review


The prevalence of paralogous enzymes implies that novel catalytic functions can evolve on preexisting protein scaffolds. The weak secondary activities of proteins, which reflect catalytic promiscuity and substrate ambiguity, are plausible starting points for this evolutionary process. In this study, we observed the emergence of a new enzyme from the ASKA collection of Escherichia coli open reading frames (ORFs). The over-expression of (His)6-tagged glutamine phosphoribosylpyrophosphate amidotransferase (PurF) unexpectedly rescued a ΔtrpF E. coli strain from starvation on minimal media. The wild-type PurF and TrpF enzymes are unrelated in sequence, tertiary structure or catalytic mechanism. The promiscuous phosphoribosylanthranilate isomerase (PRAI) activity of the ASKA PurF variant apparently stems from a pre-existing affinity for phosphoribosylated substrates. The relative fitness of the (His)6-PurF/ΔtrpF strain was improved 4.8-fold to nearly wild-type levels by random mutagenesis of purF and genetic selection. The evolved and ancestral PurF proteins were purified and reacted with phosphoribosylanthranilate in vitro. The best evolvant (kcat/KM = 0.3 s−1.M−1) was ~25-fold more efficient than its ancestor, but >107–fold less efficient than the wild-type PRAI. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms.

Copyright information:

© 2008 Elsevier Ltd. All rights reserved.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported License (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Creative Commons License

Export to EndNote