Abstract Evidence suggests that novel enzyme functions evolved from low-level promis- cuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems- level adaptations are poorly understood.
Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promis- cuity in the acquisition and optimization of growth on predicted non-native substrates in E. After as few as 20 generations, the evolving populations repeatedly acquired the capacity to grow on five pre- dicted novel substrates—D-lyxose, Ddeoxyribose, D-arabinose, m-tartrate, Email address: afeist ucsd.
Promiscuous enzyme activities played key roles in multiple phases of adaptation. Altered promiscuous activities not only established novel high- efficiency pathways, but also suppressed undesirable metabolic routes. Fur- ther, structural mutations shifted enzyme substrate turnover rates towards the new substrate while retaining a preference for the primary substrate. Fi- nally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity.
Computational approaches will be essential to synthesize the complex role of promiscuous activities in applied biotechnology and in models of evolutionary adaptation. Keywords: adaptive evolution, enzyme promiscuity, systems biology, genome-scale modeling 1 1. Introduction 2 Understanding how novel metabolic pathways arise during adaptation to 3 environmental changes remains a central issue in evolutionary biology.
The 4 prevailing view is that enzymes often display promiscuous i. However, it 7 remains poorly explored how and at what evolutionary stages enzyme side 8 activities contribute to environmental adaptations. Innovations 13 have been linked to beneficial mutations that endow an organism with novel 14 capabilities such as the ability to use a new carbon source and expand into 15 a new ecological niche[11, 12]. This is distinct from optimizations associated 16 with mutations that improve upon the initial innovation.
It is often observed 17 that the mutations accrued within this optimization phase produce gradual 18 benefits in fitness[11]. Typically, enzyme promiscuity has been linked to the 19 innovation phase, for which mutations enhancing secondary activities may 20 result in dramatic phenotypic improvements[2, 11]. In this work, we demon- 21 strate that enzyme promiscuity can be linked to fitness benefits in both the 22 innovation and optimization stages of adaptive evolution.
There has been an increasing interest 26 in studying empirical fitness landscapes to assess the predictability of evolu- 27 tionary routes[15, 16]. However, these approaches assess predictability only 28 in retrospect and there is a need for computational frameworks that forecast 29 the specific genes that accumulate mutations based on mechanistic knowl- 30 edge of the evolving trait.
However, it re- 34 mains unclear whether this approach could predict evolution in a population 35 of cells adapting to a new nutrient environment through spontaneous muta- 36 tions. First, phenotypes conferred by artificial overexpression might not be 37 accessible through single mutations arising spontaneously.
Second, and more 38 fundamentally, mutations in distinct genes may lead to the same phenotype. Furthermore, computational approaches can aid in predicting 41 and discovering overlapping physiological functions of enzymes [15, 18], but 42 these have also yet to be explored in the context of adaptation.
In this study, 43 we address these issues by performing controlled laboratory evolution exper- 44 iments to adapt E. Results and Discussion 47 2. Computational prediction and experimental evolution of non-native car- 48 bon source utilizations 49 To test our ability to predict evolutionary adaptation to novel non- 50 native carbon sources based on our knowledge of underground metabolism, 51 we utilized a comprehensive network reconstruction of underground metabolism[4].
By adding the set of underground reactions 55 to the comprehensive metabolic reconstruction for E. Duplicate laboratory evolution ex- 71 periments were conducted in batch growth conditions and in parallel on an 72 automated adaptive laboratory evolution ALE platform using a protocol 73 that uniquely selected for adaptation to conditions where the ancestor i.
In the innovation phase, E. A description of the complex passage protocol 77 is given in the Figure 1 legend and expanded in the methods for both phases 78 of the evolution. This procedure successfully adapted E.
Unsuccessful cases could 81 be attributed to various experimental and biological factors such as exper- 82 imental duration limitations, the requirement of multiple mutation events, 83 or stepwise adaptation events, as observed in an evolving E. Underground metabolism accurately predicted the genes mutated during 86 innovation 87 To analyze the mutations underlying the nutrient utilizations, clones were 88 isolated and sequenced shortly after an innovative growth phenotype was 89 achieved and mutations were identified see Methods and analyzed for their 90 associated causality Fig.
S1, Dataset S1. Strong signs of par- 91 allel evolution were observed at the level of mutated genes in the replicate 92 evolution experiments. Such parallelism provided evidence of the beneficial 93 nature of the observed mutations and is a prerequisite for predicting the 94 genetic basis of adaptation[22]. Not only were the specific genes or their direct regulatory elements mutated in four out of five cases, but few additional mutations per strain, Dataset S1 were observed in the initial innovation phase, indicating that the innovations required a small number mutational steps to activate the predicted growth phenotype and the method utilized was highly selective.
For the one case where the prediction and observed mutations did not align, D-arabinose, a detailed inspection of the literature revealed existing evidence that three fuc operon associated en- zymes can metabolize D-arabinose—FucI, FucK, and FucA [23]. In this case, the modeling approach could not make the correct prediction because the promiscuous underground reaction database was incomplete.
Of the fifteen mutation events outlined in Table 1, eleven were categorized as regulatory observed in all five suc- cessful substrate conditions and four were categorized as structural three of five successful substrate conditions.
For D-lyxose, Ddeoxyribose, and m-tartrate evolution experiments, mutations were observed within the cod- ing regions of the predicted genes, namely yihS, rbsK, and dmlA Table 1, Figs. Regulatory mutations, occurring in transcriptional regu- lators or within intergenic regions—likely affecting sigma factor binding and transcription of the predicted gene target—were observed for D-lyxose, D- 2-deoxyribose, m-tartrate, and monomethyl succinate Table 1.
Observing more regulatory mutations is broadly consistent with previous reports[10, 24]. Fur- thermore, although enzyme dosage could also be increased through dupli- cation of genomic segments, this scenario was not commonly observed dur- ing the innovation phase of our experiments. Furthermore, the large duplication in the Ddeoxyribose strain could not be reconstructed using this method due to the limitations of the method. Individual mutants were isolated after pORT- MAGE reconstruction, and their growth was monitored on the innovative growth medium over the course of one week.
The growth test revealed that single mutations were sufficient for growth on D-lyxose, D-arabinose, and m-tartrate, but with varying lengths of time for growth to be detected de- pending on the mutation present Table S3. Overall, these causality assessments support the notion that underground activities open short adaptive paths towards novel phenotypes. The N20Y sole mutation observed in the RbsK enzyme during the evolution on Ddeoxyribose served as a case study.
Previous work has found that predominantly intestinal and ex- traintestinal strains of E. Specifi- cally, four such reported pathogenic strains in this set three E. This information suggests that the N20Y mutation may have improved the ribokinase underground activity of RbsK in the mutant strain evolved here on Ddeoxyribose and enabled growth in this environment similar to the capabilities of the strains that possess the DeoK enzyme see Fig.
S5 for a structural comparison. Contribution of enzyme side activities to the optimization phase of adap- tation Once the roles of mutations acquired during the innovation phase were es- tablished, adaptive mechanisms required for optimizing or fine-tuning growth on the novel carbon sources were explored.
Analysis of mutations in the opti- mization phase led to identification of additional promiscuous enzyme activi- ties, above and beyond the innovative mechanisms, impacting the phenotypes of the evolved strains in four of the five nutrient conditions Table 2. Dis- covery of these optimizing activities was driven by a systems-level analysis consisting of mutation, enzyme activity, and transcriptome analyses coupled with computational modeling of optimized growth states on the novel carbon sources.
Marked and repeatable in- creases in growth rates on the non-native carbon sources was observed in as few as generations Table S1. Such plateaus represent re- gions where a causal mutation has fixed in a population[20]. Out of the total set of 41 mutations identified in the growth optimization regimes Datasets S1, S2 , a subset Table 2 was explored.
This subset consisted of genes which were repeatedly mutated in replicate experiments or across all endpoint se- quencing data on a given carbon source. To unveil the potential mechanisms for improving growth on the non-native substrates, the transcriptome of ini- tial and endpoint populations right after the innovation phase and at the end of the optimization phase, respectively was analyzed using RNAseq.
Additionally, for the D-lyxose experiments, enzyme activity was analyzed to determine the effect of a structural mutations acquired in a key enzyme during growth optimization. Protein structural mutations were observed beyond those ob- served during after the initial innovation. Structural mutations are believed to improve the enzyme side activity to achieve the optimization, and this effect was experimentally verified.
The effects of structural mutations on enzyme activity were examined for the YihS isomerase enzyme that was mu- tated during the D-lyxose evolution Fig. A cell-free in vitro transcription and translation system[32, 33] was used to express the enzymes and examine conversions of D-mannose to D-fructose a primary activity[34] and D-lyxose to D-xylulose side activity Fig. The ratios of the turnover rates of D-lyxose to the turnover rates of D-mannose were cal- culated and compared Fig. Based on structural analysis of AraC Fig.
It was thus reasoned that AraB was catalyzing the conversion of D-ribulose to D-ribulose 5-phosphate in an alternate pathway for metabolizing D-arabinose Fig. This signaled the possibility of a growth advantage for using the araB enabled pathway and thus was explored experimentally. Experimental growth rate measurements of clones carrying either the fucK or araBAD genes knockouts showed that the FucK enzyme activity was essential for growth on D-arabinose for all strains ana- lyzed innovative and optimized Fig.
However, removal of araB from optimized endpoint strains reduced the growth rate of the strain to the approximate growth rate of the initial innovative strain Fig. Putting this in the context of previous work, a similar pathway has been described in mutant Klebsiella aerogens W70 strains[41]. In the study, it was suggested that the D-ribulosephosphate pathway i. This conclusion supports the role of the optimization mutations observed here in araC.
Overall, underground activities of both the fuc operon innovative mutations and ara operon optimizing mutations encoded enzymes were important for the adaptation to efficiently metabo- lize D-arabinose and the ara mutated operon did not solely support growth. Two additional proposed mechanisms for growth optimization on m-tartrate and D-lyxose were related to the primary activities of pyrE and xylB and are discussed in the Supporting Text Fig.
Loss of an enzyme side activity improves fitness Analysis of the Ddeoxyribose adaptation revealed a conceptually novel way by which alterations in promiscuous enzyme activities contribute to growth optimization. Several lines of observation suggested that suppres- sion of a side reaction of aldehyde dehydrogenase A AldA enhanced growth on this novel carbon source. The optimizing mutation event in the D deoxyribose evolution was a large deletion event spanning genes Fig.
Of these, the metabolic gene that was most significantly expressed in the ancestor i. AldA has been described as a broad substrate specificity enzyme and has shown cat- alytic activity on acetaldehyde[42].
Turning to computational modeling to understand the impact of an active AldA, showed that forcing increased flux through acetaldehyde to acetate conversion decreased the overall growth rate Fig. Together, these findings indicate that the large deletion event observed in the Ddeoxyribose endpoint selected against the AldA side activity, leading to improved growth.
This scenario suggests that not only enhancement, but also suppression of side reactions can play a piv- otal role in adaptation to novel environments. Conclusions Taken together, the results of this combined computational analysis and laboratory evolution study show that enzyme promiscuity is prevalent in metabolism and plays a major role in both phenotypic innovation and op- timization during adaptation.
It was demonstrated that enzyme side activ- ities can confer a fitness benefit in two distinct ways. First, side activities contributed to the establishment of novel metabolic routes that enabled or improved the utilization of a new nutrient source.
Second, suppression of an undesirable underground activity that diverted flux from a newly established pathway conferred a fitness benefit. Here, it was demonstrated that computational metabolic network mod- els which include the repertoire of enzyme side activities made it possible to predict the genetic basis of adaptation to novel carbon sources.
As such, systems models and analyses are likely to contribute significantly towards representing the complex implications of promiscuity in theoretical models of molecular evolution[43]. Furthermore, the evolution of new gene functions from secondary promiscuous activities has been proposed by multiple models assuming functional gene divergence from a common ancestor following gene duplication events[44, 45, 46, 7, 47, 48] and the findings and strains from this study are relevant towards better understanding such models.
Finally, the computational and subsequent approaches developed in this work can be leveraged to understand promiscuous activity in engineered strains for industrial biotechnology and in the adaptation of pathogenic microbes. Materials and Methods Flux balance analysis and sampling in silico methods utilized in this work are described in SI Materials and Methods.
Detailed information regard- ing the laboratory evolution experiments, growth media composition, and whole genome sequencing and mutation analysis is provided in SI Materials and Methods. Furthermore, details regarding the pORTMAGE library con- struction and mutant isolation as well as the cell-free in vitro transcription, translation enzyme activity characterizations performed are also provided in SI Materials and Methods.
Experimental methods utilized to analyze opti- mization regime mechanisms of adaptation including RNA sequencing, phage transduction mutagenesis, and individual mutant growth characterizations are included in SI Materials and Methods.
Acknowledgments We would like to thank Richard Szubin and Ying Hefner for assistance with strain resequencing and P1-phage transduction mutagenesis, as well as Elizabeth Brunk for helpful discussion. Declaration of Interests The authors declare no competing interests. References [1] R. Jensen, Enzyme recruitment in evolution of new function, Annu. Khersonsky, D. Tawfik, Enzyme promiscuity: a mechanistic and evolutionary perspective, Annu. Nam, N. Lewis, J. Lerman, D. Lee, R. Chang, D.
Kim, B. Palsson, Network context and selection in the evolution to enzyme specificity, Science — Notebaart, B. Szappanos, B. Kintses, F. Bo- gos, V. Spohn, B. Wagner, E. Ruppin, C. Papp, Network-level architecture and the evolutionary potential of underground metabolism, Proc.
Huang, F. Hippauf, D. Rohrbeck, M. Haustein, K. Wenke, J. Feike, N. Sorrelle, B. The improvement in the promiscuous activity has been shown wide-ranging from to fold. This fold improvement was not correlated with the original level of the promiscuous activity.
Similarly, the decrease in the native activity varied from to fold [ 38 ]. These results suggest that evolution of a novel enzyme may be possible with some orthologous enzymes, but not with all [ 39 , 40 ]. Hence, it is suggested to use several orthologous enzymes, instead of only one, as a starting point for directed laboratory evolution. As we have discussed above, the promiscuity of enzymes can be a starting point for the divergent evolution.
The low promiscuous activity towards a physiologically irrelevant substrate might turn the enzyme into a much more proficient catalyst which is possible by accumulation of just one or more beneficial mutations affording a survival benefit to the organism. Furthermore, a promiscuous function can be the starting point for the creation of a new enzyme activity by applying rational, semi-rational protein engineering methodologies or by applying directed laboratory evolution [ 41 , 42 ].
Organophosphates OPs poisoning poses great danger to both military and civilian populations. A timely and effective control with pharmacological agents can minimize the damage.
Hydrolysis of OPs by mammalian enzymes at high efficiency has been a challenge, specially the more toxic stereoisomer Sp , with a required catalytic efficiency.
Though mammalian PON1 is a foremost candidate for such a treatment, it hydrolyzes the toxic Sp isomers of G-agents with very slow rates. We characterized several such mutants with broader stereo-specificity and also with different leaving groups, which could be used for the in vivo detoxification of real nerve agents. These in vivo prophylactic activity of evolved variants and the newly developed screens provide the foundation for designing PON1 and other OP degrading enzymes for prophylaxis against other G-type agents [ 3 , 43 ].
Improvement in promiscuous function of mammalian serum PON1. Another category of nerve agent is V-type VX and VR , which is known to be one of the most toxic substances.
Promiscuous bacterial phosphotriesterases PTEs are capable of hydrolyzing VX but with very low level of activity [ 44 ]. PTE mutant library was created by mutating 12 active-site residues of PTE in order to enhance its catalytic efficiency. The library was screened for catalytic activity against a new VX analogue, DEVX, which contains the same thiolate-leaving group of VX attached to a diethoxyphosphate center rather than the ethoxymethylphosphonate center of VX.
However, stereo-selectivity for the hydrolysis of the two enantiomers of VX was relatively low [ 44 ]. The catalytic activity of PTE from A. X-ray crystal structures for the above variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky tyrosine residue with the smaller alanine residue [ 45 ]. One example of creating highly active and specific enzyme is reported with sortase A from Staphylococcus aureus, which catalyzes the transpeptidation of an LPXTG peptide acceptor and a glycine-linked peptide donor.
This has proven to be a powerful tool for site-specific protein modification due to its transpeptidation activity but its high specificity limits its broader utility. Recently, two orthogonal sortase A variants have been developed by applying directed laboratory evolution technique that recognize each of two different substrates, LAXTG and LPXSG, with high activity and specificity.
The evolved sortases exhibit changes in specificity up to 51,fold, relative to the starting sortase without much loss of catalytic activity [ 46 ]. Lipases are well studied due to high industrial value and applications.
Candida antarctica lipase B CALB is a well-known for its promiscuous activity which can be utilized for designing new catalysts for important organic reactions [ 47 ]. The hydrolysis of the trans-diphenylpropene oxide is studied in particular and suggested that the mutant CALB is a good protein scaffold to be used for the biosynthesis of chiral compounds [ 48 ].
Similarly, lipase SrLip from Streptomyces rimosus Q93MW7 revealed lipase, phospholipase, esterase and thioesterase activities [ 49 ].
Porcine pancreatic lipase PPL , one of the best enzymes identified for biocatalytic aldol addition at lower temperature but much accelerated activity at elevated temperature. Recently, a novel peptidase is reported from thermophilic archaea Sulfolobus tokodaii for its catalytic promiscuity of aldol addition, which shows comparable activity as PPL.
This study is an example, which signifies the importance to identify a new enzyme with catalytic promiscuity and demonstrates the application of novel biocatalyst from thermophile microorganisms [ 50 ]. A recent report describes the designing of an enzyme capable of discriminately etherifying the parahydroxyl of coniferyl alcohol in the presence of excess sinapyl alcohol. The designed enzyme mutant has a considerably smaller substrate-binding pocket that forces a clear steric hindrance thus excluding larger lignin precursors.
Lignin is derivative of three monolignols, which are polymerized by oxidative reactions. The composition of the monolignol monomers determines the degree of lignin condensation and thus the degradability of plant cell walls.
Guaiacyl lignin is considered as the condensed structural unit [ 50 ]. The active site of a monolignol 4-O-methyltransferase MOMT5 was precisely remodeled to create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Together with the mutant information, crystal structural information with combinatorial active site saturation mutagenesis were applied and remodeled its substrate binding pocket by the addition of four substitutions, i.
Type I plant nucleases are known to play an important role in apoptotic processes and cell senescence. The first structure of tomato nuclease showed its oligomerization and activity profiles resulted in unexpected promiscuous activity towards phospholipids.
Solving the crystal structure of this protein identified possible binding sites for double stranded DNA and other nucleic acids. Essentially, the phospholipase activity of tomato nuclease I significantly broaden the substrate promiscuity of the enzyme, and resulted in the release of diacylglycerol. The diacylglycerol is an important second messenger that can be related to the role of tomato nuclease I in apoptosis [ 52 ]. The above observations support the hypothesis that promiscuous functions are not exceptions but inherent features of proteins in general.
However, the mechanisms of exhibiting promiscuity are different in different proteins, enzymes in particular. It can be categorized in three major groups based on the different factors responsible for enzyme promiscuity: 1 active site plasticity of enzymes, 2 substrate ambiguity and 3 cofactor ambiguity.
However, the importance of promiscuous functions especially with physiologically irrelevant substrates is yet to be understood. The promiscuous functions can also be utilized as a starting point for the evolution of new proteins in laboratory conditions for biotechnological applications. Annu Rev Biochem — Copley SD An evolutionary perspective on protein moonlighting. Biochem Soc Trans 42 6 — Nat Chem Biol 7 2 — Biochemistry 54 35 — Nat Biotechnol — Copley SD Evolution of efficient pathways for degradation of anthropogenic chemicals.
Nat Chem Biol 5 8 — Chem Rec 15 4 — Article Google Scholar. J Mol Biol 13 — Mol Biol Evol 31 6 — Trends Biochem Sci 40 2 — Atkins WM Biological messiness vs. J Steroid Biochem Mol Biol — Sustainable Chemical Processes Curr Opin Chem Biol — Biochem J 2 — BMC Evol Biol PLoS Biol 12 4 :e Antimicrob Agents Chemother — J Mol Biol 4 — Chem Bio Chem. However, as Nobeli et al. Large number of successful results which prove this are already available in the literature [ 12 ],[ 71 ]-[ 73 ].
He et al. Similarly, Baas et al. The review of Baas et al. It is interesting that all enzymes have N-terminal proline as a key catalytic residue. It is very well established that some amino acids as such catalyse diverse kinds of reactions on their own [ 76 ],[ 77 ].
Obviously, the catalytic promiscuity of enzymes has its origin in confluence of different factors [ 11 ]. Table 2 summarizes the usefulness of catalytic promiscuity in biocatalysis Table 2.
Archae constitute the oldest organisms on our earth. Given the harsher conditions prevalent in those days, archae are prominent examples of extremophiles. The specialised functions of the enzymes part of the evolved metabolism in more complex organism have evolved from the small number of enzymes which these ancestors had.
So, these organisms constitute valuable systems to track extensive promiscuity shown by the early enzymes.
Jia et al. Archae enzymes are rich in intrinsic disorder. So, promiscuity is one facet of the evolutionary design of enzymes. As Skolnick et al. The example of glutathione transferase is especially interesting. Atkins group [ 85 ] have discussed that two isoforms of glutathione transfereases in humans vastly differ in their catalytic promiscuity. This highlights the conceptual relationship between the phenomenon of isoenzymes to promiscuous behaviour as pointed out by one of us few years back [ 5 ].
On the other hand, GSTA acted upon lipid peroxidation products. More recent work from the same group [ 86 ] concludes that smooth barrier free transitions within the local conformational landscape of the active site is associated with the more promiscuous GST. Furthermore, local molten globule behaviour optimizes the catalytic function of the GST in detoxification [ 87 ]. Its active site is quite different from cytosolic GST reported so far. It may be interesting to see the unravelling of its specificity in the years to come.
Finally, as pointed out by them, GSTs are also possibly involved in the storage and transportation of wide variety of biological molecules and thus are also moonlighting proteins. This three way correlation between isoenzymes, promiscuous enzymes and moonlighting proteins have been pointed out earlier [ 5 ]. To sum up, the conformational pliability may be local or global in protein structure as the cause behind promiscuity. Few trends are clear.
While during early few years, examples of catalytic promiscuity were mostly concerned with applications of lipases; last few years have seen other classes of enzymes other hydrolases, oxidoreductases, transferases etc. The shift from our belief in enzyme specificity to the realization that these biocatalysts are fairly promiscuous has not been gradual. Some key milestones can be identified. We accepted the idea of broad specificity lately called relaxed specificity long ago.
Isoforms or isoenzymes, the enzymes from the same organism, carrying similar catalytic activity but with different specificity and kinetic behaviour have been again known since several decades [ 5 ]. The binding site, part of the active centre was known to show promiscuous behaviour when textile dyes emerged as powerful affinity ligands [ 89 ]. This is akin to a general practitioner becoming a specialist in medical science but retaining enough knowledge of how to treat many diseases.
In the beginning, we had RNA world [ 90 ]. Then came early enzymes. As complex metabolisms were required with evolution of more complex organisms, more specialised enzymes emerged. These enzymes did not forget entirely what their ancestors were capable of. Hopefully, it will draw attention of many biotechnologists to look at the huge iceberg of potential application of these green catalysts to nurture sustainable approaches in chemical industries.
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