ORCID Profile
0000-0002-8235-1829
Current Organisation
University of British Columbia
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Publisher: eLife Sciences Publications, Ltd
Date: 05-02-2019
DOI: 10.7554/ELIFE.40789
Abstract: Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may impact the evolvability of proteins, but the underlying molecular basis remains unclear. Here, we performed comparative directed evolution of four orthologous metallo-β-lactamases toward a new function and found that different starting genotypes evolved to distinct evolutionary outcomes. Despite a low initial fitness, one ortholog reached a significantly higher fitness plateau than its counterparts, via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show how cryptic molecular properties and conformational variation of active site residues in the initial genotypes cause epistasis, that could lead to distinct evolutionary outcomes. Our work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution.
Publisher: Elsevier BV
Date: 06-2018
DOI: 10.1016/J.SBI.2017.09.005
Abstract: This review focuses on recent work that has begun to establish specific functional roles for protein conformational dynamics, specifically how the conformational landscapes that proteins can s le can evolve under laboratory based evolutionary selection. We discuss recent technical advances in computational and biophysical chemistry, which have provided us with new ways to dissect evolutionary processes. Finally, we offer some perspectives on the emerging view of conformational dynamics and evolution, and the challenges that we face in rationally engineering conformational dynamics.
Publisher: Springer Science and Business Media LLC
Date: 23-04-2018
DOI: 10.1038/S41589-018-0043-2
Abstract: The emergence of enzymes through the neofunctionalization of noncatalytic proteins is ultimately responsible for the extraordinary range of biological catalysts observed in nature. Although the evolution of some enzymes from binding proteins can be inferred by homology, we have a limited understanding of the nature of the biochemical and biophysical adaptations along these evolutionary trajectories and the sequence in which they occurred. Here we reconstructed and characterized evolutionary intermediate states linking an ancestral solute-binding protein to the extant enzyme cyclohexadienyl dehydratase. We show how the intrinsic reactivity of a desolvated general acid was harnessed by a series of mutations radiating from the active site, which optimized enzyme-substrate complementarity and transition-state stabilization and minimized s ling of noncatalytic conformations. Our work reveals the molecular evolutionary processes that underlie the emergence of enzymes de novo, which are notably mirrored by recent ex les of computational enzyme design and directed evolution.
Publisher: Elsevier
Date: 2020
Publisher: Springer Science and Business Media LLC
Date: 24-02-2022
DOI: 10.1038/S41559-022-01675-5
Abstract: Fitness landscapes, mappings of genotype henotype to their effects on fitness, are invaluable concepts in evolutionary biochemistry. Although widely discussed, measurements of phenotype-fitness landscapes in proteins remain scarce. Here, we quantify all single mutational effects on fitness and phenotype (EC
Publisher: Cold Spring Harbor Laboratory
Date: 15-04-2021
DOI: 10.1101/2021.04.14.439889
Abstract: Fitness landscapes, mappings of genotype henotype to their effects on fitness, are invaluable concepts in evolutionary biochemistry. Though widely discussed, measurements of phenotype-fitness landscapes in proteins remain scarce. Here, we quantify all single mutational effects on fitness and phenotype of VIM-2 β-lactamase across a 64-fold range of icillin concentrations. We then construct a phenotype-fitness landscape that takes variations in environmental selection pressure into account. We found that a simple, empirical landscape accurately models the ~39,000 mutational data points, suggesting the evolution of VIM-2 can be predicted based on the selection environment. Our landscape provides new quantitative knowledge on the evolution of the β-lactamases and proteins in general, particularly their evolutionary dynamics under sub-inhibitory antibiotic concentrations, as well as the mechanisms and environmental dependence of nonspecific epistasis. An empirical fitness landscape discloses the environmental dependence of mutational effects in VIM-2 β-lactamase.
Publisher: Cold Spring Harbor Laboratory
Date: 20-02-2020
DOI: 10.1101/2020.02.19.956706
Abstract: Metallo-β-lactamases (MBLs) degrade a broad spectrum of β-lactam antibiotics, and are a major disseminating source for multidrug resistant bacteria. Despite many biochemical studies in erse MBLs, molecular understanding of the roles of residues in the enzyme’s stability and function, and especially substrate specificity, is lacking. Here, we employ deep mutational scanning (DMS) to generate comprehensive single amino acid variant data on a major clinical MBL, VIM-2, by measuring the effect of thousands of VIM-2 mutants on the degradation of three representative classes of β-lactams ( icillin, cefotaxime, and meropenem) and at two different temperatures (25°C and 37°C). We revealed residues responsible for expression and translocation, and mutations that increase resistance and/or alter substrate specificity. The distribution of specificity-altering mutations unveiled distinct molecular recognition of the three substrates. Moreover, these function-altering mutations are frequently observed among naturally occurring variants, suggesting that the enzymes has continuously evolved to become more potent resistance genes.
Publisher: eLife Sciences Publications, Ltd
Date: 08-06-2020
DOI: 10.7554/ELIFE.56707
Abstract: Metallo-β-lactamases (MBLs) degrade a broad spectrum of β-lactam antibiotics, and are a major disseminating source for multidrug resistant bacteria. Despite many biochemical studies in erse MBLs, molecular understanding of the roles of residues in the enzyme’s stability and function, and especially substrate specificity, is lacking. Here, we employ deep mutational scanning (DMS) to generate comprehensive single amino acid variant data on a major clinical MBL, VIM-2, by measuring the effect of thousands of VIM-2 mutants on the degradation of three representative classes of β-lactams ( icillin, cefotaxime, and meropenem) and at two different temperatures (25°C and 37°C). We revealed residues responsible for expression and translocation, and mutations that increase resistance and/or alter substrate specificity. The distribution of specificity-altering mutations unveiled distinct molecular recognition of the three substrates. Moreover, these function-altering mutations are frequently observed among naturally occurring variants, suggesting that the enzymes have continuously evolved to become more potent resistance genes.
Publisher: Springer Science and Business Media LLC
Date: 12-06-2020
Publisher: Elsevier BV
Date: 03-2019
DOI: 10.1016/J.JMB.2019.01.041
Abstract: Genetic variation among orthologous genes has been largely formed through neutral genetic drift while maintaining the functional role of these genes. However, because the evolution of gene occurs in the context of each host organism, their sequence changes are also associated with adaptation to a specific environment. Thus, genetic variation can create critical phenotypic variation, particularly when genes are transferred to a new host by horizontal gene transfer. Unveiling "hidden phenotypic variation" is particularly important for genes that confer resistance to antibiotics. However, our understanding of the molecular mechanisms that underlie phenotypic variation remains limited. Here we sought to determine the extent of phenotypic variation in the B1 metallo-β-lactamase (MBL) family and its molecular basis by systematically characterizing eight MBL orthologs, including NDM-1 and VIM-2 and IMP-1. We found that these MBLs confer erse levels of resistance. The phenotypic variation cannot be explained by variation in catalytic efficiency alone rather, it is the combination of the catalytic efficiency and abundance of functional periplasmic enzyme that best predicts the observed variation in resistance. The level of functional periplasmic expression varied dramatically between MBL orthologs. This was the result of changes at multiple levels of each ortholog's: (1) quantity of mRNA, (2) amount of MBL expressed, and (3) efficacy of functional enzyme translocation to the periplasm. Overall, it is the interaction between each gene and the host's underlying cellular processes (transcription, translation, and translocation) that determines MBL genetic incompatibility through horizontal gene transfer. These host-specific processes may constrain the effective spread and deployment of MBLs to certain host species and could explain the current observed distribution bias.
Publisher: Cold Spring Harbor Laboratory
Date: 26-12-2018
DOI: 10.1101/504811
Abstract: Characterizing the adaptive landscapes that encompass the emergence of novel enzyme functions can provide molecular insights into both enzymatic and evolutionary mechanisms. Here, we combine ancestral protein reconstruction with biochemical, structural, and mutational analyses to characterize the functional evolution of methyl-parathion hydrolase (MPH), a xenobiotic organophosphate-degrading enzyme. We identify five mutations that are necessary and sufficient for the evolution of MPH from an ancestral dihydrocoumarin hydrolase. In-depth analyses of the adaptive landscapes encompassing this evolutionary transition revealed that a complex interaction network, defined in part by higher-order epistasis, determined the adaptive pathways that were available. By also characterizing the adaptive landscapes in terms of their functional activity towards three other OP substrates, we reveal that subtle differences in substrate substituents drastically alter the enzyme’s epistatic network by changing its intramolecular interactions. Our work suggests that the mutations function collectively to enable substrate recognition via subtle structural repositioning.
Publisher: Springer Science and Business Media LLC
Date: 05-04-2019
DOI: 10.1038/S41467-019-09534-X
Abstract: Cofactor F 420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F 420 has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho- l -lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho- l -lactate, as the substrate, leading to the formation of the previously uncharacterized intermediate dehydro-F 420 -0. The C-terminal domain of FbiB then utilizes FMNH 2 to reduce dehydro-F 420 -0, which produces mature F 420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F 420 from a recombinant F 420 biosynthetic pathway in Escherichia coli .
Publisher: eLife Sciences Publications, Ltd
Date: 27-12-2018
Publisher: Cold Spring Harbor Laboratory
Date: 15-11-2018
DOI: 10.1101/470336
Abstract: Cofactor F 420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F 420 has not been fully eluci-dated: neither the enzyme that generates the putative intermediate 2-phospho- L -lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we show that the guanylyltransferases FbiD and CofC accept phosphoenolpyruvate, rather than 2-phospho- L -lactate, as their substrate, leading to the formation of the previously uncharacterized intermediate, dehydro-F 420 -0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F 420 -0, which produces mature F 420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. This new insight has allowed the heterologous expression F 420 from a recombinant F 420 biosynthetic pathway in Escherichia coli .
Publisher: American Society for Microbiology
Date: 27-10-2020
Abstract: There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a erse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.
Publisher: Cold Spring Harbor Laboratory
Date: 06-04-2023
DOI: 10.1101/2023.04.05.535609
Abstract: Phenotypic variations within a population exist on different scales of biological organization and play a central role in evolution by providing adaptive capacity at the population-level. Thus, the question of how evolution generates phenotypic variation within an evolving population is fundamental in evolutionary biology. Here we address this question by performing experimental evolution of an antibiotic resistance gene, VIM-2 β-lactamase, combined with erse biochemical assays and population genetics. We found that neutral drift, i.e. , evolution under a static environment, with a low antibiotic concentration can promote and maintain significant phenotypic variation within the population with -fold differences in resistance strength. We developed a model based on the phenotype-environment-fitness landscape generated with ,000 VIM-2 variants, and demonstrated that the combination of “mutation-selection balance” and “threshold-like fitness-phenotype relationship” is sufficient to explain the generation of large phenotypic variation within the evolving population. Importantly, high-resistance conferring variants can emerge during neutral drift, without being a product of adaptation. Our findings provide a novel and simple mechanistic explanation for why most genes in nature, and by extension, systems and organisms, inherently exhibit phenotypic variation, and thus, population-level evolvability.
Publisher: Oxford University Press (OUP)
Date: 2021
Abstract: β-Lactamases represent one of the most prevalent resistance mechanisms against β-lactam antibiotics. Beyond their clinical importance, they have also become key models in enzymology and evolutionary biochemistry. A global understanding of their evolution and sequence and functional ersity can therefore aid a wide set of different disciplines. Interestingly, β-lactamases have evolved multiple times from distinct evolutionary origins, with ancestries that reach back billions of years. It is therefore no surprise that these enzymes exhibit erse structural features and enzymatic mechanisms. In this review, we provide a bird’s eye view on the evolution of β-lactamases within the two enzyme superfamilies—i.e. the penicillin-binding protein-like and metallo-β-lactamase superfamily—through phylogenetics. We further discuss potential evolutionary origins of each β-lactamase class by highlighting signs of evolutionary connections in protein functions between β-lactamases and other enzymes, especially cases of enzyme promiscuity.
Publisher: eLife Sciences Publications, Ltd
Date: 05-06-2020
Publisher: Springer Science and Business Media LLC
Date: 02-02-2019
DOI: 10.1038/S41589-019-0386-3
Abstract: Characterizing the adaptive landscapes that encompass the emergence of novel enzyme functions can provide molecular insights into both enzymatic and evolutionary mechanisms. Here, we combine ancestral protein reconstruction with biochemical, structural and mutational analyses to characterize the functional evolution of methyl-parathion hydrolase (MPH), an organophosphate-degrading enzyme. We identify five mutations that are necessary and sufficient for the evolution of MPH from an ancestral dihydrocoumarin hydrolase. In-depth analyses of the adaptive landscapes encompassing this evolutionary transition revealed that the mutations form a complex interaction network, defined in part by higher-order epistasis, that constrained the adaptive pathways available. By also characterizing the adaptive landscapes in terms of their functional activities towards three additional organophosphate substrates, we reveal that subtle differences in the polarity of the substrate substituents drastically alter the network of epistatic interactions. Our work suggests that the mutations function collectively to enable substrate recognition via subtle structural repositioning.
No related grants have been discovered for Nobuhiko Tokuriki.