Two-step Ligand Binding in a (βα)8 Barrel Enzyme

Söderholm, Annika; Guo, Xiaohu; Newton, Matilda; Evans, Gary B.; Näsvall, Joakim; Patrick, Wayne M.; Selmer, M.

doi:10.1074/jbc.m115.678086

Cited by 18 publications

(32 citation statements)

References 31 publications

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“…As can be seen in Figure 4 , the most common mutations were located in or near the active site. For example, mutations in positions 80, 81, 83, and 102, which interact with the phosphate adjoining the non-reacting ribose (phosphate 2) of ProFAR ( Söderholm et al, 2015 ) were common, as were mutations in the nearby positions 79, 106, and 107. Without structures and enzyme kinetic data of the different mutant proteins it is challenging to explain how they can confer their effect but some observations can be made.…”

Section: Resultsmentioning

confidence: 99%

“…For example, the phosphate 2 binding site ( Figures 4C,D ) is likely to be important for proper positioning of the native substrate ProFAR, and mutations in nearby positions could alter the phosphate binding site to allow PRA binding ( Newton et al, 2017 ). Purified wt HisA coordinates PO 4 2– or SO 4 2– in both phosphate binding sites ( Söderholm et al, 2015 ), and the presence of a negatively charged ion in this site causes electrostatic repulsion of the negatively charged PRA, thus preventing binding of PRA and TrpF activity. Mutations affecting the phosphate 2 binding site and reducing the binding of PO 4 2– and SO 4 2– would likely increase binding of PRA, improving TrpF activity with a collateral loss of HisA activity.…”

Section: Resultsmentioning

confidence: 99%

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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%

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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“…tuberculosis (mtPriA), the catalytic efficiencies of tmHisA, ddHisA, and pcHisA are about tenfold higher ( Table 1 , HisA reaction). They are comparable to the catalytic efficiency of HisA from Salmonella enterica (seHisA), which is considered to be an archetypical representative of the HisA family [ 12 ]. Strikingly, tmHisA, ddHisA, and pcHisA also displayed TrpF-activity, something that has not been shown before.…”

Section: Resultsmentioning

confidence: 99%

“…Although the exact reasons for this discrepancy are unknown, it may be due to differences in enzyme purification and handling. 4 Data taken from [ 12 ]; n. d.: values were not determined. 5 n. d.: k cat and could not be determined individually; was deduced from the linear part of the saturation curve.…”

Section: Resultsmentioning

confidence: 99%

Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation

et al. 2016

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Modern enzymes are highly optimized biocatalysts that process their substrates with extreme efficiency. Many enzymes catalyze more than one reaction; however, the persistence of such ambiguities, their consequences and evolutionary causes are largely unknown. As a paradigmatic case, we study the history of bi-functionality for a time span of approximately two billion years for the sugar isomerase HisA from histidine biosynthesis. To look back in time, we computationally reconstructed and experimentally characterized three HisA predecessors. We show that these ancient enzymes catalyze not only the HisA reaction but also the isomerization of a similar substrate, which is commonly processed by the isomerase TrpF in tryptophan biosynthesis. Moreover, we found that three modern-day HisA enzymes from Proteobacteria and Thermotogae also possess low TrpF activity. We conclude that this bi-functionality was conserved for at least two billion years, most likely without any evolutionary pressure. Although not actively selected for, this trait can become advantageous in the case of a gene loss. Such exaptation is exemplified by the Actinobacteria that have lost the trpF gene but possess the bi-functional HisA homolog PriA, which adopts the roles of both HisA and TrpF. Our findings demonstrate that bi-functionality can perpetuate in the absence of selection for very long time-spans.

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“…For all mutation effect predictors that required a crystal structure to be used, the structure of Salmonella enterica HisA(D7N, D176A) with ProFAR (PDB ID: 5A5W ( Söderholm et al. 2015 )) was used.…”

Section: Methodsmentioning

confidence: 99%

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA

Lundin

Tang

Guy

et al. 2017

Molecular Biology and Evolution

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The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients.

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Two-step Ligand Binding in a (βα)8 Barrel Enzyme

Cited by 18 publications

References 31 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

Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA

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