Interconverting the Catalytic Activities of (βα)8-barrel Enzymes from Different Metabolic Pathways: Sequence Requirements and Molecular Analysis

Leopoldseder, Sonja; Claren, Jörg; Jürgens, Catharina; Sterner, Reinhard

doi:10.1016/j.jmb.2004.01.062

Cited by 45 publications

(58 citation statements)

References 37 publications

(47 reference statements)

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“…Remarkably, the alternative conformation of loop ␤6␣6 allows for the formation of a hydrogen bond between an oxygen atom of the substrate phosphate group and the backbone nitrogen of glycine 170 (bond length, 3.06 Å). Also, we note that the crucial exchanges D127V and D169V both lead to the removal of a negative charge, which might facilitate binding of the negatively charged PRA due to relief of electrostatic repulsion (14).…”

Section: Pra (µM)mentioning

confidence: 89%

“…The TrpF activities of the purified HisAF and HisA variants were followed at 25°C by a fluorimetric assay and analyzed as described (14,25). The binding of the product analogue rCdRP to HisAFcomIII and HisA-II was followed by fluorescence energy transfer and analyzed as described (15,31).…”

Section: Methodsmentioning

confidence: 99%

“…Along these lines, we earlier used random mutagenesis and selection in vivo to establish the catalytic activity of phosphoribosyl anthranilate (PRA) isomerase (TrpF) on the (␤␣) 8 -barrel scaffold of NЈ-[(5Ј-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA) from Thermotoga maritima (13,14). The wild-type TrpF and HisA enzymes catalyze mechanistically related isomerisation reactions of aminoaldoses into the corresponding aminoketoses within tryptophan and histidine biosynthesis (15).…”

mentioning

confidence: 99%

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Establishing wild-type levels of catalytic activity on natural and artificial (βα) ₈ -barrel protein scaffolds

Claren

Malisi²,

Höcker³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The generation of high levels of new catalytic activities on natural and artificial protein scaffolds is a major goal of enzyme engineering. Here, we used random mutagenesis and selection in vivo to establish a sugar isomerisation reaction on both a natural (␤␣) 8-barrel enzyme and a catalytically inert chimeric (␤␣) 8-barrel scaffold, which was generated by the recombination of 2 (␤␣) 4-half barrels. The best evolved variants show turnover numbers and substrate affinities that are similar to those of wild-type enzymes catalyzing the same reaction. The determination of the crystal structure of the most proficient variant allowed us to model the substrate sugar in the novel active site and to elucidate the mechanistic basis of the newly established activity. The results demonstrate that natural and inert artificial protein scaffolds can be converted into highly proficient enzymes in the laboratory, and provide insights into the mechanisms of enzyme evolution.chimeric protein ͉ enzyme design ͉ enzyme evolution ͉ half barrel ͉ TIM barrel

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Section: Pra (µM)mentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Establishing wild-type levels of catalytic activity on natural and artificial (βα) ₈ -barrel protein scaffolds

Claren

Malisi²,

Höcker³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…They showed that single point mutations can impart PRAI activity on the HisA protein, which catalyzes an analogous Amadori rearrangement, 26 and also on HisF, which normally catalyzes the subsequent synthesis of the imidazole ring. 27 Here, we searched the ASKA library for a promiscuous PRAI, and then enhanced its activity by directed evolution. We describe the structure and function of an alternative scaffold for PRAI activity, which began as a completely unrelated enzyme from the de novo purine biosynthesis pathway.…”

Section: Introductionmentioning

confidence: 99%

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

Patrick

Matsumura

2008

Journal of Molecular Biology

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SummaryThe 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 (k cat /K M = 0.3 s −1 .M −1 ) was ~25-fold more efficient than its ancestor, but >10 7 -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.

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“…In particular, similarities in sequence, structure, and function suggest that several (␤␣) 8 -barrels from the tryptophan and histidine biosynthetic pathways have evolved divergently from a common ancestral enzyme (10,11). In support of this hypothesis, both NЈ [(5Ј-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (ProFAR) isomerase (HisA) and imidazole glycerol phosphate synthase (HisF), which catalyze two consecutive reactions of histidine biosynthesis, could be converted by exchanging a single amino acid into enzymes with phosphoribosylanthranilate isomerase (TrpF) activity (12,13).…”

mentioning

confidence: 99%

Mimicking enzyme evolution by generating new (βα) ₈ -barrels from (βα) ₄ -half-barrels

Höcker

Claren

Sterner

2004

Proc. Natl. Acad. Sci. U.S.A.

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Gene duplication and fusion events that multiply and link functional protein domains are crucial mechanisms of enzyme evolution. The analysis of amino acid sequences and three-dimensional structures suggested that the (␤␣) 8-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (␤␣) 4-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (␤␣) 8-barrels from (␤␣) 4-half-barrels that were deduced from the enzymes imidazole glycerol phosphate synthase (HisF) and N [(5 -phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide isomerase (HisA). To this end, the gene for the C-terminal (␤␣) 4-half-barrel (HisF-C) of HisF was duplicated and fused in tandem to yield HisF-CC, which is more stable than HisF-C. In the next step, by optimizing side-chain interactions within the center of the ␤-barrel of HisF-CC, the monomeric and compact (␤␣) 8-barrel protein HisF-C*C was generated. Moreover, the genes for the N-and C-terminal (␤␣) 4-half-barrels of HisF and HisA were fused crosswise to yield the chimeric proteins HisFA and HisAF. Whereas HisFA contains native secondary structure elements but adopts ill-defined association states, the (␤␣) 8-barrel HisAF is a stable and compact monomer that reversibly unfolds with high cooperativity. The results obtained suggest a previously undescribed dimension for the diversification of enzymatic activities: new (␤␣) 8-barrels with novel functions might have evolved by the exchange of (␤␣) 4-halfbarrel domains with distinct functional properties. chimeric proteins ͉ gene duplication ͉ histidine biosynthesis ͉ TIM-barrel

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Interconverting the Catalytic Activities of (βα)8-barrel Enzymes from Different Metabolic Pathways: Sequence Requirements and Molecular Analysis

Cited by 45 publications

References 37 publications

Establishing wild-type levels of catalytic activity on natural and artificial (βα) ₈ -barrel protein scaffolds

Establishing wild-type levels of catalytic activity on natural and artificial (βα) ₈ -barrel protein scaffolds

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

Mimicking enzyme evolution by generating new (βα) ₈ -barrels from (βα) ₄ -half-barrels

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Interconverting the Catalytic Activities of (βα)8-barrel Enzymes from Different Metabolic Pathways: Sequence Requirements and Molecular Analysis

Cited by 45 publications

References 37 publications

Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

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

Mimicking enzyme evolution by generating new (βα) 8 -barrels from (βα) 4 -half-barrels

Contact Info

Product

Resources

About

Establishing wild-type levels of catalytic activity on natural and artificial (βα) ₈ -barrel protein scaffolds

Establishing wild-type levels of catalytic activity on natural and artificial (βα) ₈ -barrel protein scaffolds

Mimicking enzyme evolution by generating new (βα) ₈ -barrels from (βα) ₄ -half-barrels