Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 377, Issue 2, Pages 323-336Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.01.043
Keywords
catalytic promiscuity; contingency; directed evolution; enzyme evolution; multicopy suppressor
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Funding
- NIGMS NIH HHS [R01 GM074264, R01 GM074264-03] Funding Source: Medline
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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 (A Complete Set of E. coli K-12 ORF Archive) collection of Escherichia coli open reading frames. The overexpression of (His)(6)-tagged glutamine phosphoribosylpyrophosphate amiclotransferase (PurF) unexpectedly rescued a A trpF E. coli strain from starvation on minimal media. The wild-type PurF and TrpF enzymes are unrelated in sequence, tertiary structure and catalytic mechanism. The promiscuous phosphoribosylanthranilate isomerase activity of the ASKA PurF variant apparently stems from a preexisting affinity for phosphoribosylated substrates. The relative fitness of the (HiS)(6)-PurF/ Delta trpF 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 (k(cat)/K-M=0.3 s(-1) M-1) was similar to 25-fold more efficient than its ancestor but > 10(7) -fold less efficient than the wild-type phosphoribosylanthranilate isomerase. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms. (C) 2008 Elsevier Ltd. All rights reserved.
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