Functional transitions in enzyme evolution: Balancing stability, folding and catalytic specificity

Evolutionary pathways by which proteins have evolved in Nature over billions of years have resulted in an impressive diversity of structures that carry out many functions with unrivalled efficiency. Directed protein evolution in the test tube can emulate natural evolution, but is often limited by low hit rates and small improvements during evolutionary cycles. Furthermore, the combination of mutations that is needed for large improvements cannot always be reached by one-by-one mutational steps due to the occurrence of general loss-of-function mutations or epistatic ratchets. The question then arises how evolutionary dead ends can be avoided. Important parameters that shape these fitness landscapes are e.g. expression level, stability and catalytic activity/specificity. We are currently probing these parameters for ancestral sequences inferred from phylogenetic relationships between members of the catalytically diverse metallo-β-lactamase1 and alkaline phosphatase2-4 superfamilies. Mapping of substrate specificity profiles on the genetic relationships allowed the identification of the ancestral nodes between which transitions in primary function most likely occur. The latter is one of the key processes in evolution of new functions. The substrate specificity profiles of the current enzymes suggest that the change in primary function is the result of a shift in substrate preference rather than de novo evolutionary invention of a novel activity. Furthermore several characterized ancestral sulfatases suggest a shift toward increased specificity over evolutionary time, as well as a trend that ancestral enzymes are more stable than present day enzymes. References 1 Baier & Tokuriki (2014) Connectivity between catalytic landscapes of the metallo-β-lactamase superfamily. J. Mol. Biol. 426, 2442-2456. 2 Jonas & Hollfelder (2009) Mapping catalytic promiscuity in the alkaline phosphatase superfamily. Pure. Appl. Chem. 81, 731-742. 3 van Loo et al. (2010) An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily, Proc. Natl. Acad. Sci. U. S. A. 107, 2740-2745. 4 van Loo et al (2017) Balancing specificity and promiscuity in enzyme superfamily evolution: multidimensional activity transitions. submitted

Gene content evolution in the arthropods

Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity

Gene content evolution in the arthropods

Background: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. Results: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. Conclusions: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity

The modular nature of protein evolution: domain rearrangement rates across eukaryotic life

Abstract Background Modularity is important for evolutionary innovation. The recombination of existing units to form larger complexes with new functionalities spares the need to create novel elements from scratch. In proteins, this principle can be observed at the level of protein domains, functional subunits which are regularly rearranged to acquire new functions. Results In this study we analyse the mechanisms leading to new domain arrangements in five major eukaryotic clades (vertebrates, insects, fungi, monocots and eudicots) at unprecedented depth and breadth. This allows, for the first time, to directly compare rates of rearrangements between different clades and identify both lineage specific and general patterns of evolution in the context of domain rearrangements. We analyse arrangement changes along phylogenetic trees by reconstructing ancestral domain content in combination with feasible single step events, such as fusion or fission. Using this approach we explain up to 70% of all rearrangements by tracing them back to their precursors. We find that rates in general and the ratio between these rates for a given clade in particular, are highly consistent across all clades. In agreement with previous studies, fusions are the most frequent event leading to new domain arrangements. A lineage specific pattern in fungi reveals exceptionally high loss rates compared to other clades, supporting recent studies highlighting the importance of loss for evolutionary innovation. Furthermore, our methodology allows us to link domain emergences at specific nodes in the phylogenetic tree to important functional developments, such as the origin of hair in mammals. Conclusions Our results demonstrate that domain rearrangements are based on a canonical set of mutational events with rates which lie within a relatively narrow and consistent range. In addition, gained knowledge about these rates provides a basis for advanced domain-based methodologies for phylogenetics and homology analysis which complement current sequence-based methods

Complex Regulatory Role of DNA Methylation in Caste- and Age-Specific Expression of a Termite

The reproductive castes of eusocial insects are often characterized by extreme lifespans and reproductive output, indicating an absence of the fecundity/longevity trade-off. The role of DNA methylation in the regulation of caste- and age-specific gene expression in eusocial insects is controversial. While some studies find a clear link to caste formation in honeybees and ants, others find no correlation when replication is increased across independent colonies. Although recent studies have identified transcription patterns involved in the maintenance of high reproduction throughout the long lives of queens, the role of DNA methylation in the regulation of these genes is unknown. We carried out a comparative analysis of DNA methylation in the regulation of caste-specific transcription and its importance for the regulation of fertility and longevity in queens of the higher termite Macrotermes natalensis . We found evidence for significant, well-regulated changes in DNA methylation in mature compared to young queens, especially in several genes related to ageing and fecundity in mature queens. We also found a strong link between methylation and caste-specific alternative splicing. This study reveals a complex regulatory role of fat body DNA methylation both in the division of labour in termites, and during the reproductive maturation of queens

Adaptive landscapes and molecular basis for the functional innovation of an organophosphate hydrolysing enzyme

How functional innovation of enzyme, i.e., the acquirement of novel functions, occurs is a fundamental question in molecular evolution. Here, we unveil the evolutionary processes that led to the emergence of methyl parathion hydrolase (MPH), an enzyme that has acquired the ability to degrade the xenobiotic organophosphate (OP), methyl-parathion (Pt-M). Combining ancestral sequence reconstruction (ASR) with biochemical, genetic, and structural analyses, we characterized the adaptive landscape of MPH during its functional transition towards Pt-M activity from a dihydrocoumarin (DHC)-degrading ancestor. Five amino acid substitutions were found to be critical for the conversion between the ancestral DHC activity and the evolved Pt-M activity, accounting for a >600,000-fold functional switch between the two substrates. Characterization of adaptive landscapes of 5 combinatorial mutational space (32 combinations) revealed that the prevalence of epistatic interactions between the residues; consequently, only a fraction (16) of the 120 (5!) possible pathways are actually accessible in the gradually incremental manner. Moreover, multiple adaptive landscapes analysis for four similar OP compounds unveiled molecular basis for the development of high substrate specificity toward Pt-M over other compounds. Our study provides a comprehensive description of the evolutionary and molecular mechanisms that led to the emergence of a novel enzymatic function

Higher-order epistasis shapes the fitness landscape of a xenobiotic-degrading enzyme

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.N.T. and E.B.-B. thank the Human Frontier Science Program (HFSP) for support via research grant RGP0006/2013. N.T. acknowledges support by the Natural Sciences and Engineering Research Council of Canada (NSERC) via discovery grants RGPIN 418262-12 and RGPIN 2017-04909. N.T. is a CIHR new investigator and a Michael Smith Foundation of Health Research (MSFHR) career investigator. S.C.L.K. thanks the Knut and Alice Wallenberg Foundation (Wallenberg Academy Fellowships 2013.0124 and 2018.0140) and the Swedish National Infrastructure for Computing (SNIC). D.W.A. thanks NSERC and the MSFHR for post-doctoral support