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Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study.

Background The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. Results About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Conclusion Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html .

This article is dedicated to the memory of Michael G. Rossmann. Dating back to the last universal common ancestor, P-loop NTPases and Rossmanns comprise the most ubiquitous and diverse enzyme lineages. Despite similarities in their overall architecture and phosphate binding motif, a lack of sequence identity and some fundamental structural differences currently designates them as independent emergences. We systematically searched for structure and sequence elements shared by both lineages. We detected homologous segments that span the first βαβ motif of both lineages, including the phosphate binding loop and a conserved aspartate at the tip of β2. The latter ligates the catalytic metal in P-loop NTPases, while in Rossmanns it binds the nucleotide’s ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the β2-Asp to take either one of these two roles. While convergence cannot be completely ruled out, we show that both lineages likely emerged from a common βαβ segment that comprises the core of these enzyme families to this very day.

Key messageThis review outlines research performed in the last two decades on the structural, kinetic, regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzyme for starch biosynthesis.ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.

Abstract How does enzymatic activity emerge? To shed light on this fundamental question, we study type B dihydrofolate reductases (DfrB), which were discovered for their role in antibiotic resistance. These rudimentary enzymes are evolutionarily distinct from the ubiquitous, monomeric FolA dihydrofolate reductases targeted by the antibiotic trimethoprim. DfrB is unique: it homotetramerizes to form a highly symmetrical central tunnel that accommodates its substrates in close proximity and the right orientation, thus promoting the metabolically essential production of tetrahydrofolate. It is the only known enzyme built from the ancient Src Homology 3 fold, typically a binding module. Strikingly, by studying the evolution of this enzyme family, we observe that no active-site residues are conserved across catalytically active homologs. Integrating experimental and computational analyses, we identify an intricate relationship between homotetramerization and catalytic activity, where formation of a tunnel featuring positive electrostatic potential proves to be a powerful predictor of activity. We demonstrate that the DfrB enzymes have not evolved in response to the synthetic antibiotic to which they confer strong resistance, and propose that DfrB domains evolved the capacity for rudimentary catalysis from a binding capacity. That (rudimentary) catalysis can emerge from the homotetramerization of a binding domain, and that it has been recently recruited by pathogenic bacteria, manifests the opportunistic nature of evolution.

Terpenoids constitute a large and diverse class of natural products that serve many functions in nature. Most of the tens of thousands of the discovered terpenoids are synthesized by plants, where they function as primary metabolites involved in growth and development, or as secondary metabolites that optimize the interaction between the plant and its environment. Several plant terpenoids are economically important molecules that serve many applications as pharmaceuticals, pesticides, etc. Major challenges for the commercialization of plant-derived terpenoids include their low production levels in planta and the continuous demand of industry for novel molecules withnewor superior biological activities. Here, wehighlight several synthetic biology methods to enhance and diversify the production of plant terpenoids, with a foresight towards triterpenoid engineering, the least engineered class of bioactive terpenoids. Increased or cheaper production of valuable triterpenoids may be obtained by ‘classic’ metabolic engineering of plants or by heterologous production of the compounds in other plants or microbes. Novel triterpenoid structures can be generated through combinatorial biosynthesis or directed enzyme evolution approaches. In its ultimate form, synthetic biology may lead to the production of large amounts of plant triterpenoids in in vitro systems or custom-designed artificial biological systems.

Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an α/β folding pattern with a central beta sheet flanked by 2 - 3 α-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

Enzymes speed up reactions that would otherwise be too slow to sustain the metabolism of selfreplicators. Yet, most enzymes seem only moderately efficient, exhibiting kinetic parameters orders of magnitude lower than their expected physically achievable maxima and spanning over surprisingly large ranges of values. Here, we question how these parameters evolve using a mechanistic model where enzyme efficiency is a key component of individual competition for resources. We show that kinetic parameters are under strong directional selection only up to a point, above which enzymes appear to evolve under near-neutrality, thereby confirming the qualitative observation of other modeling approaches. While the existence of a large fitness plateau could potentially explain the extensive variation in enzyme features reported, we show using a population genetics model that such a widespread distribution is an unlikely outcome of evolution on a common landscape, as mutation–selection–drift balance occupy a narrow area even when very moderate biases towards lower efficiency are considered. Instead, differences in the evolutionary context encountered by each enzyme should be involved, such that each evolves on an individual, unique landscape. Our results point to drift and effective population size playing an important role, along with the kinetics of nutrient transporters, the tolerance to high concentrations of intermediate metabolites, and the reversibility of reactions. Enzyme concentration also shapes selection on kinetic parameters, but we show that the joint evolution of concentration and efficiency does not yield extensive variance in evolutionary outcomes when documented costs to protein expression are applied.

Abstract Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the “tip of the iceberg” since thousands of sequences remain uncharacterized in this enzyme family alone.

Radiation of the plant pyridoxal 5′-phosphate (PLP)-dependent aromatic L-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic L-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehydederived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)- norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.