Directed evolution of (  )8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase  -subunit

Evran, Serap; Telefoncu, Azmi; Sterner, Reinhard

doi:10.1093/protein/gzs015

Cited by 15 publications

(15 citation statements)

References 49 publications

(47 reference statements)

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“…Of these, about 1% (15/1457) could confer TrpF function. This suggests that there are many different pathways toward obtaining a new activity within HisA, illustrating the versatility of the (βα) 8 -barrel fold to acquire novel activities ( Jespersen et al, 1993 ; Fong et al, 2000 ; Wierenga, 2001 ; Henn-Sax et al, 2002 ; Schmidt et al, 2003 ; Vega et al, 2003 ; Sterner and Höcker, 2005 ; Li et al, 2008 ; Evran et al, 2012 ).…”

Section: Resultsmentioning

confidence: 99%

Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence

2020

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When a new activity evolves by changes in a pre-existing enzyme this is likely to reduce the original activity, generating a functional trade-off. The properties of this trade-off will affect the continued evolution of both functions. If the trade-off is strong, gene duplication and subsequent divergence would be favored whereas if the trade-off is weak a bi-functional enzyme could evolve that performs both functions. We previously showed that when a bi-functional HisA enzyme was evolved under selection for both HisA and TrpF functions, evolution mainly proceeded via duplication-divergence and specialization, implying that the trade-off is strong between these two functions. Here, we examined this hypothesis by identifying the mutational pathways (i.e., the mutational landscape) in the Salmonella enterica HisA enzyme that conferred a TrpF-like activity, and examining the trade-offs between the original and new activity. For the HisA enzyme there are many different paths toward the new TrpF function, each with its own unique trade-off. A total of 16 single mutations resulted in HisA enzyme variants that acquired TrpF activity and only three of them maintained HisA activity. Twelve mutants were evolved further toward increased TrpF activity and during evolution toward improved TrpF activity the original HisA activity was completely lost in all lineages. We propose that, aside from various relevant ecological factors, two main genetic factors influence whether evolution of a new function proceeds via duplication – divergence (specialization) or by evolution of a generalist: (i) the relative mutation supply of the two pathways and (ii) the shape of the trade-off curve between the native and new function.

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Section: Resultsmentioning

confidence: 99%

Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence

2020

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“…The N-terminal domain consists of three anti-parallel β-strands, followed by four α-helices that are connected by the flexible loop L1 (residues 38–45). The C-terminal domain contains the widely occurring 8-stranded β/α barrel architecture [ 44 , 45 ]. The barrel, however, does not contain the typical (βα) 8 fold as found in triosephosphate isomerase (TIM)-barrel enzymes [ 46 ], but rather comprises a β 2 α 2 (βα) 6 fold that is also seen in enolases from yeast or other bacterial origins [ 15 , 47 , 48 ].…”

Section: Resultsmentioning

confidence: 99%

Stability of the Octameric Structure Affects Plasminogen-Binding Capacity of Streptococcal Enolase

et al. 2015

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Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation.

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“…Nevertheless, HisA can gain specificity for the TrpF substrate, PRA, after random gain-of-function mutations selected for in a Δ trpF background [1,3]. Moreover, PRA specificity has been found to be highly evolvable in other mechanistically unrelated protein folds [4,5]. This poses PRA isomerase activity as a suitable enzymatic function to investigate functional shifts involving co-occurring analogous enzymes.…”

Section: Introductionmentioning

confidence: 99%

Co-occurrence of analogous enzymes determines evolution of a novel (βα)8-isomerase sub-family after non-conserved mutations in flexible loop

Verduzco-Castro

Michalska

Endres

et al. 2016

Biochemical Journal

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We investigate the evolution of co-occurring analogous enzymes involved in L-tryptophan and L-histidine biosynthesis in Actinobacteria Phylogenetic analysis of trpF homologues, a missing gene in certain clades of this lineage whose absence is complemented by a dual-substrate HisA homologue, termed PriA, found that they fall into three categories: (i) trpF-1, an L-tryptophan biosynthetic gene horizontally acquired by certain Corynebacterium species; (ii) trpF-2, a paralogue known to be involved in synthesizing a pyrrolopyrrole moiety and (iii) trpF-3, a variable non-conserved orthologue of trpF-1 We previously investigated the effect of trpF-1 upon the evolution of PriA substrate specificity, but nothing is known about the relationship between trpF-3 and priA After in vitro steady-state enzyme kinetics we found that trpF-3 encodes a phosphoribosyl anthranilate isomerase. However, mutation of this gene in Streptomyces sviceus did not lead to auxothrophy, as expected from the biosynthetic role of trpF-1 Biochemical characterization of a dozen co-occurring TrpF-2 or TrpF-3, with PriA homologues, explained the prototrophic phenotype, and unveiled an enzyme activity trade-off between TrpF and PriA. X-ray structural analysis suggests that the function of these PriA homologues is mediated by non-conserved mutations in the flexible L5 loop, which may be responsible for different substrate affinities. Thus, the PriA homologues that co-occur with TrpF-3 represent a novel enzyme family, termed PriB, which evolved in response to PRA isomerase activity. The characterization of co-occurring enzymes provides insights into the influence of functional redundancy on the evolution of enzyme function, which could be useful for enzyme functional annotation.

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Directed evolution of ( )8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase -subunit

Cited by 15 publications

References 49 publications

Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence

Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence

Stability of the Octameric Structure Affects Plasminogen-Binding Capacity of Streptococcal Enolase

Co-occurrence of analogous enzymes determines evolution of a novel (βα)8-isomerase sub-family after non-conserved mutations in flexible loop

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