A study in molecular contingency: glutamine phosphoribosylpyrophosphate amidotransferase is a promiscuous and evolvable phosphoribosylanthranilate isomerase |
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Authors: | Patrick Wayne M Matsumura Ichiro |
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Institution: | 1 Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University, Atlanta, GA 30322, USA 2 Institute of Molecular Biosciences, Massey University, Auckland 0745, New Zealand |
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Abstract: | 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 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 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/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 phosphoribosylanthranilate isomerase. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms. |
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Keywords: | AcTrpD anthranilate phosphoribosyltransferase enzyme from Acinetobacter baylyi strain ADP1 ASKA A Complete Set of E coli K-12 ORF Archive Cam chloramphenicol CdRP 1&prime -(2&prime -carboxyphenylamino)-1&prime -deoxyribulose 5&prime -phosphate epPCR error-prone PCR GFP green fluorescent protein HPLC high-performance liquid chromatography IGPS indoleglycerol phosphate synthase InGP indoleglycerol phosphate IPTG d-thiogalactoside" target="_blank">isopropyl-β-d-thiogalactoside Kan kanamycin ORF open reading frame PRA phosphoribosylanthranilate PRAI phosphoribosylanthranilate isomerase PRPP 5-phosphoribosyl-1-pyrophosphate PurF glutamine phosphoribosylpyrophosphate amidotransferase R5P ribose-5-phosphate PRTase phosphoribosyltransferase |
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