Adventures on the routes of enzyme evolution – In memoriam Dan Tawfik

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Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily.

Insight regarding how diverse enzymatic functions and reactions have evolved from ancestral scaffolds is fundamental to understanding chemical and evolutionary biology, and for the exploitation of enzymes for biotechnology. We undertook an extensive computational analysis using a unique and comprehensive combination of tools that include large-scale phylogenetic reconstruction to determine the sequence, structural, and functional relationships of the functionally diverse flavin mononucleotide-dependent nitroreductase (NTR) superfamily (>24,000 sequences from all domains of life, 54 structures, and >10 enzymatic functions). Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a minimal flavin-binding scaffold. We identified the structural design principle for this divergence: Insertions at key positions in the minimal scaffold that, combined with the fixation of key residues, have led to functional specialization. These results will aid future efforts to delineate the emergence of functional diversity in enzyme superfamilies, provide clues for functional inference for superfamily members of unknown function, and facilitate rational redesign of the NTR scaffold

Enzyme evolution and engineering using insertions and deletions

In Nature, proteins evolve and acquire new functions by accumulating mutations. Substitutions and InDels (Insertions and Deletions), as well as circular permutations and rearrangement of protein domains, account for the majority of evolutionary changes. While the effects of substitutions have been extensively studied and documented, understanding the structural and functional effects of InDels still remains a challenge. InDels are assumed to be highly deleterious mutations because they are more likely to disrupt the structural integrity of proteins than are substitutions. On the other hand, they may induce significant structural changes that substitutions alone cannot cause and thus are believed to be key players in many natural evolutionary processes, such as the modification of active site loops to generate new enzyme functions1 or the emergence of new protein structures2. We aimed at performing directed evolution by randomly incorporating InDels to investigate how they would be tolerated and whether they could be selected for functional improvements. Starting from a previously reported methodology3, we developed a library construction approach to randomly incorporate InDels within a DNA sequence of interest and applied it to generate InDel variant libraries of a promiscuous enzyme (phosphotriesterase4). We screened the resulting libraries (i) to compare the impact of InDels to that of substitutions on the enzyme, (ii) to identify adaptive InDels improving a new (or promiscuous) activity and (iii) to investigate the interaction between InDels and substitutions in an adaptive process. Our results show that, while being generally more deleterious than substitutions, InDels can also lead to functional improvements and may allow access to alternative evolutionary trajectories. References 1 Park et al. (2006). Science 311, 535–538. 2 Grishin (2001). J Struct Biol 134:167–185. 3 Jones et al. (2014). Methods in Mol Biol 1179:159-72. 4 Tokuriki et al. (2012). Nature Comm 3:1257

Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, Discovery Grant RGPIN 418262- 12, http://www.nserc-crsng.gc.ca/), the Biotechnology and Biological Sciences Research Council (BBSRC, Grant BB/L002469/1, http://www. bbsrc.ac.uk/), the European Research Council (ERC, Advanced Investigator Grant 695669, https:// erc.europa.eu/), and the Human Frontiers Science Program (Grant RGP0006/2013, http://www.hfsp. org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Thrice upon a time: The repeated emergence of a novel enzymatic function from an evolvable protein scaffold

Understanding the emergence of new protein functions from their ancestors is a long-standing challenge in biology and biotechnology; many questions remain unanswered. How can one protein scaffold support multiple distinct functions? How are diverse functions of a superfamily connected? How are major functional switches achieved? Large-scale experimental approaches that systematically determine the activity profiles across enzyme superfamilies have now begun to provide comprehensive views of functional diversity and evolutionary relationships. Intriguing insights can be gained: promiscuous activities are prevalent and many divergent proteins retain functional connectivity via enzyme promiscuity1.Interested in the varied biological and biotechnological roles of FMN-dependent “nitroreductase” enzymes (NTRs), we undertook extensive computational and functional analyses to determine sequence, structural and functional relationships2. This large and diverse superfamily contains \u3e80,000 sequences from all domains of life, 54 structures, and \u3e10 enzymatic functions. Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a highly “evolvable” minimal flavin-binding scaffold. To investigate the diverse NTR sequence space for the capacity to catalyze nitroreduction, we synthesized \u3e500 genes and performed high-throughput activity screening to profile 18 in vivo substrates. In vitro kinetic analysis was subsequently performed on 112 enzymes against 32 substrates (vs. 2 nicotinamide cofactors), equating to \u3e7,000 reactions3. We demonstrated that only four of the 22 distinct superfamily subgroups display canonical nitroaromatic reductase activities. Eight additional subgroups display occasional promiscuous activities with selected substrates, and 10 subgroups display no nitroreductase activity. Structural analyses revealed the underlying molecular details: nitroreduction has emerged three distinct times in the superfamily via three unique molecular solutions - loop insertions at three different positions in the NTR scaffold, combined with the fixation of key residues, have independently led to functional specialization. These results are now facilitating the rational redesign of the NTR scaffold. Our work provides clues for functional inference for sequences of unknown function, and will aid future efforts to exploit evolvable scaffolds for engineering, and understand the emergence of functional diversity in enzyme superfamilies. Baier F, Copp JN, Tokuriki N. Biochemistry. 2016 Nov 22;55(46):6375-6388. Akiva E*, Copp JN*, Tokuriki N, Babbitt PC. Proc Natl Acad Sci U S A. 2017 114(45):E9549-E9558. Copp JN, Morales DM, Chang S, Jiang K, Akiva E, Babbitt PC, Tokuriki N. in prep

Primary cutaneous signet ring cell carcinoma expressing cytokeratin 20 immunoreactivity

Primary cutaneous signet ring cell carcinoma (PCSRCC) is a very unusual but distinctive clinicopathologic entity that can simulate metastatic adenocarcinomas. It is defined as a diffuse malignant epithelial neoplasia localized in the dermis and subcutis without epidermal involvement, showing variable amounts of signet ring cells, without evidence of visceral adenocarcinoma. We present 2 cases of PCSRCC, which involved eyelids and axilla respectively. Despite thorough systemic workup, primary sources could not be demonstrated in either case. The tumor cells are positive for gross cystic disease fluid protein 15 in addition to a variety of glandular markers. Furthermore, both cases were immunostained with cytokeratin 20 (CK20). In conclusion, we report 2 cases of PCSRCC expressing CK20 immunoreactivity. CK20-positive primary cutaneous tumors should include PCSRCC in addition to Merkel cell carcinoma

Evolution of β-lactamase-mediated cefiderocol resistance

Background: Cefiderocol is a novel siderophore β-lactam with improved hydrolytic stability toward β-lactamases, including carbapenemases, achieved by combining structural moieties of two clinically efficient cephalosporins, ceftazidime and cefepime. Consequently, cefiderocol represents a treatment alternative for infections caused by MDR Gram-negatives. Objectives: To study the role of cefiderocol on resistance development and on the evolution of β-lactamases from all Ambler classes, including KPC-2, CTX-M-15, NDM-1, CMY-2 and OXA-48. Methods: Directed evolution, using error-prone PCR followed by selective plating, was utilized to investigate how the production and the evolution of different β-lactamases cause changes in cefiderocol susceptibility determined using microbroth dilution assays (MIC and IC50). Results: We found that the expression of blaOXA-48 did not affect cefiderocol susceptibility. On the contrary, the expression of blaKPC-2, blaCMY-2, blaCTX-M-15 and blaNDM-1 substantially reduced cefiderocol susceptibility by 4-, 16-, 8- and 32-fold, respectively. Further, directed evolution on these enzymes showed that, with the acquisition of only 1–2 non-synonymous mutations, all β-lactamases were evolvable to further cefiderocol resistance by 2- (NDM-1, CTX-M-15), 4- (CMY-2), 8- (OXA-48) and 16-fold (KPC-2). Cefiderocol resistance development was often associated with collateral susceptibility changes including increased resistance to ceftazidime and ceftazidime/ avibactam as well as functional trade-offs against different β-lactam drugs. Conclusions: The expression of contemporary β-lactamase genes can potentially contribute to cefiderocol resistance development and the acquisition of mutations in these genes results in enzymes adapting to increasing cefiderocol concentrations. Resistance development caused clinically important cross-resistance, especially against ceftazidime and ceftazidime/avibactam

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

Optimization processes, such as evolution, are constrained by diminishing returns - the closer the optimum, the smaller the benefit per mutation, and by tradeoffs - improvement of one property at the cost of others. However, the magnitude and molecular basis of these parameters, and their effect on evolutionary transitions, remain unknown. Here we pursue a complete functional transition of an enzyme with a >109-fold change in the enzyme's selectivity using laboratory evolution. We observed strong diminishing returns, with the initial mutations conferring >25-fold higher improvements than later ones, and asymmetric tradeoffs whereby the gain/loss ratio of the new/old activity decreased 400-fold from the beginning of the trajectory to its end. We describe the molecular basis for these phenomena and suggest they have an important role in shaping natural proteins. These findings also suggest that the catalytic efficiency and specificity of many natural enzymes may be far from their optimum

Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer

Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole L-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in L-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA’s substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in L-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism

A biophysical protein folding model accounts for most mutational fitness effects in viruses

Fitness effects of mutations fall on a continuum ranging from lethal to deleterious to beneficial. The distribution of fitness effects (DFE) among random mutations is an essential component of every evolutionary model and a mathematical portrait of robustness. Recent experiments on five viral species all revealed a characteristic bimodal shaped DFE, featuring peaks at neutrality and lethality. However, the phenotypic causes underlying observed fitness effects are still unknown, and presumably thought to vary unpredictably from one mutation to another. By combining population genetics simulations with a simple biophysical protein folding model, we show that protein thermodynamic stability accounts for a large fraction of observed mutational effects. We assume that moderately destabilizing mutations inflict a fitness penalty proportional to the reduction in folded protein, which depends continuously on folding free energy (\Delta G). Most mutations in our model affect fitness by altering \Delta G, while, based on simple estimates, \approx10% abolish activity and are unconditionally lethal. Mutations pushing \Delta G>0 are also considered lethal. Contrary to neutral network theory, we find that, in mutation/selection/drift steady-state, high mutation rates (m) lead to less stable proteins and a more dispersed DFE, i.e. less mutational robustness. Small population size (N) also decreases stability and robustness. In our model, a continuum of non-lethal mutations reduces fitness by \approx2% on average, while \approx10-35% of mutations are lethal, depending on N and m. Compensatory mutations are common in small populations with high mutation rates. More broadly, we conclude that interplay between biophysical and population genetic forces shapes the DFE.Comment: Main text: 12 pages, 5 figures Supplementary Information: 10 pages, 5 figure