Differential Effects of a Mutation on the Normal and Promiscuous Activities of Orthologs: Implications for Natural and Directed Evolution

Khanal, Akhil; McLoughlin, Sean Yu; Kershner, Jamie P.; Copley, Shelley D.

doi:10.1093/molbev/msu271

Cited by 68 publications

(68 citation statements)

References 23 publications

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“…Compared with the other bifunctional enzymes, its kinetic parameters were relatively poor for both reactions (Table 1), yet it conferred substantial growth advantages on S. enterica cells grown in the absence of histidine, or in the absence of both histidine and tryptophan (SI Appendix, Table S1). Similarly complex relationships between enzyme activity and organismal fitness were observed when bifunctional variants of ProA were evolved to become responsible for steps in both proline and arginine biosynthesis (12). Perhaps significantly, HisA(dup13-15/D10G/G102A/G11D/G44E) had the highest measured K M for the HisA substrate, ProFAR (K M ProFAR = 100 μM; Table 1).…”

Section: Resultsmentioning

confidence: 84%

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

Newton

Guo

Söderholm

et al. 2017

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

107

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New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (βα) 8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (βα) 8 barrel as an inherently evolvable protein scaffold.HisA | TrpF | adaptive evolution | enzyme performance threshold

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Section: Resultsmentioning

confidence: 84%

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

Newton

Guo

Söderholm

et al. 2017

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

107

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“…coli ProA (␥-glutamyl phosphate reductase), which catalyzes the second step in the pathway for synthesis of proline, has an inefficient promiscuous ability to catalyze the reduction of Nacetyl glutamyl phosphate (2,3). This reaction, normally catalyzed by ArgC, is essential for biosynthesis of arginine.…”

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confidence: 99%

“…However, a mutation that changes Glu383 to Ala in ProA allows the ⌬argC strain to grow, albeit slowly, on glucose (3). We previously found that E383A ProA (ProA*) has improved activity with the substrate for ArgC but substantially poorer activity with its normal substrate (2). (For technical reasons, both reactions were assayed in the reverse direction, but decreased activity in one direction implies decreased activity in the other, as well.)…”

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confidence: 99%

A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate

et al. 2016

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When microbes are faced with an environmental challenge or opportunity, preexisting enzymes with promiscuous secondary activities can be recruited to provide newly important functions. Mutations that increase the efficiency of a new activity often compromise the original activity, resulting in an inefficient bifunctional enzyme. We have investigated the mechanisms by which growth of Escherichia coli can be improved when fitness is limited by such an enzyme, E383A ProA (ProA*). ProA* can serve the functions of both ProA (required for synthesis of proline) and ArgC (required for synthesis of arginine), albeit poorly. We identified four genetic changes that improve the growth rate by up to 6.2-fold. Two point mutations in the promoter of the proBA* operon increase expression of the entire operon. Massive amplification of a genomic segment around the proBA* operon also increases expression of the entire operon. Finally, a synonymous point mutation in the coding region of proB creates a new promoter for proA*. This synonymous mutation increases the level of ProA* by 2-fold but increases the growth rate by 5-fold, an ultrasensitive response likely arising from competition between two substrates for the active site of the inefficient bifunctional ProA*. IMPORTANCEThe high-impact synonymous mutation we discovered in proB is remarkable for two reasons. First, most polar effects documented in the literature are detrimental. This finding demonstrates that polar effect mutations can have strongly beneficial effects, especially when an organism is facing a difficult environmental challenge for which it is poorly adapted. Furthermore, the consequence of the synonymous mutation in proB is a 2-fold increase in the level of ProA* but a disproportionately large 5.1-fold increase in growth rate. While ultrasensitive responses are often found in signaling networks and genetic circuits, an ultrasensitive response to an adaptive mutation has not been previously reported. Metabolic enzymes, although prodigious catalysts, are not perfectly specific for their physiological substrates. They typically possess secondary activities as a consequence of the assemblage of highly reactive functional groups, metal ions, and cofactors in their active sites. Secondary activities that are physiologically irrelevant, either because they are too inefficient to contribute to fitness or because the enzyme never encounters the substrate, are termed promiscuous activities (1).Promiscuous activities are important from an evolutionary standpoint because they provide a reservoir of catalytic potential within a proteome that can be drawn upon when the environment changes. A promiscuous activity may become important for fitness when a new source of carbon, nitrogen, or phosphorous appears in the environment or when a previously available compound, such as an amino acid or cofactor, becomes unavailable. A promiscuous activity may also become critical when the organism is exposed to a novel toxin, such as an antibiotic or pesticide.A newly recruited pro...

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“…This fold improvement was not correlated with the original level of the promiscuous activity. Similarly, the decrease in the native activity varied from 190-to 2100-fold [38]. These results suggest that evolution of a novel enzyme may be possible with some orthologous enzymes, but not with all [39,40].…”

Section: Evolutionary Potential Of Promiscuous Functionmentioning

confidence: 83%

“…Thus, the levels and the evolvability of promiscuous activities may vary among these orthologous proteins. For example, there are nine gammaglutamyl phosphate reductase (ProA) orthologs, which display different level of promiscuity varying by about 50-fold [38]. A single amino acid change from glutamine to alanine near the active site seemed to be critical for enhanced promiscuous activity in these orthologous genes.…”

Section: Evolutionary Potential Of Promiscuous Functionmentioning

confidence: 99%

Recent advances in enzyme promiscuity

Gupta

2016

Sustain Chem Process

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Enzyme promiscuity is defined as the capability of an enzyme to catalyze a reaction other than the reaction for which it has been specialized. Although, enzyme is known for its specificity, many enzymes are reported to be promiscuous in nature. However, the promiscuous function may not be relevant in physiological conditions. The reasons could be either very low level of catalytic activity or unavailability of the substrates in the cell. Hitherto, the enzyme promiscuity is of great importance because they are the starting point for the evolution of new functions in the nature. In addition, the promiscuous activities are utilized for the development of new catalytic functions by applying directed laboratory evolution and protein engineering techniques. The aim of this review is to provide recent developments on the understanding of the mechanism of catalytic promiscuity, evolvability of promiscuous functions and the applications of enzyme promiscuity in the designing of enhanced or new functional biocatalysts.

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Differential Effects of a Mutation on the Normal and Promiscuous Activities of Orthologs: Implications for Natural and Directed Evolution

Cited by 68 publications

References 23 publications

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate

Recent advances in enzyme promiscuity

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Differential Effects of a Mutation on the Normal and Promiscuous Activities of Orthologs: Implications for Natural and Directed Evolution

Cited by 68 publications

References 23 publications

Structural and functional innovations in the real-time evolution of new (βα) 8 barrel enzymes

Structural and functional innovations in the real-time evolution of new (βα) 8 barrel enzymes

A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate

Recent advances in enzyme promiscuity

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Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes

Structural and functional innovations in the real-time evolution of new (βα) ₈ barrel enzymes