EFI-EST, EFI-GNT, and EFI-CGFP: Enzyme Function Initiative (EFI) Web Resource for Genomic Enzymology Tools.

The Enzyme Function Initiative (EFI) provides a web resource with "genomic enzymology" web tools to leverage the protein (UniProt) and genome (European Nucleotide Archive; ENA; https://www.ebi.ac.uk/ena/) databases to assist the assignment of in vitro enzymatic activities and in vivo metabolic functions to uncharacterized enzymes (https://efi.igb.illinois.edu/). The tools enable (1) exploration of sequence-function space in enzyme families using sequence similarity networks (SSNs; EFI-EST), (2) easy access to genome context for bacterial, archaeal, and fungal proteins in the SSN clusters so that isofunctional families can be identified and their functions inferred from genome context (EFI-GNT); and (3) determination of the abundance of SSN clusters in NIH Human Metagenome Project metagenomes using chemically guided functional profiling (EFI-CGFP). We describe enhancements that enable SSNs to be generated from taxonomy categories, allowing higher resolution analyses of sequence-function space; we provide examples of the generation of taxonomy category-specific SSNs.

RadicalSAM.org: A Resource to Interpret Sequence-Function Space and Discover New Radical SAM Enzyme Chemistry.

The radical SAM superfamily (RSS), arguably the most functionally diverse enzyme superfamily, is also one of the largest with ~700K members currently in the UniProt database. The vast majority of the members have uncharacterized enzymatic activities and metabolic functions. In this Perspective, we describe RadicalSAM.org, a new web-based resource that enables a user-friendly genomic enzymology strategy to explore sequence-function space in the RSS. The resource attempts to enable identification of isofunctional groups of radical SAM enzymes using sequence similarity networks (SSNs) and the genome context of the bacterial, archaeal, and fungal members provided by genome neighborhood diagrams (GNDs). Enzymatic activities and in vivo functions frequently can be inferred from genome context given the tendency for genes of related function to be clustered. We invite the scientific community to use RadicalSAM.org to (i) guide their experimental studies to discover new enzymatic activities and metabolic functions, (ii) contribute experimentally verified annotations to RadicalSAM.org to enhance the ability to predict novel activities and functions, and (iii) provide suggestions for improving this resource.

Epoxyqueuosine Reductase QueH in the Biosynthetic Pathway to tRNA Queuosine Is a Unique Metalloenzyme.

Queuosine is a structurally unique and functionally important tRNA modification, widely distributed in eukaryotes and bacteria. The final step of queuosine biosynthesis is the reduction/deoxygenation of epoxyqueuosine to form the cyclopentene motif of the nucleobase. The chemistry is performed by the structurally and functionally characterized cobalamin-dependent QueG. However, the queG gene is absent from several bacteria that otherwise retain queuosine biosynthesis machinery. Members of the IPR003828 family (previously known as DUF208) have been recently identified as nonorthologous replacements of QueG, and this family was renamed QueH. Here, we present the structural characterization of QueH from Thermotoga maritima. The structure reveals an unusual active site architecture with a [4Fe-4S] metallocluster along with an adjacent coordinated iron metal. The juxtaposition of the cofactor and coordinated metal ion predicts a unique mechanism for a two-electron reduction/deoxygenation of epoxyqueuosine. To support the structural characterization, in vitro biochemical and genomic analyses are presented. Overall, this work reveals new diversity in the chemistry of iron/sulfur-dependent enzymes and novel insight into the last step of this widely conserved tRNA modification.

Evolution of Enzyme Function and the Development of Catalytic Efficiency: Triosephosphate Isomerase, Jeremy R. Knowles, and W. John Albery.

Every reader knows that an enzyme accelerates a reaction by reducing the activation-energy barrier. However, understanding how this is achieved by the structure of the enzyme and its interactions with stable complexes and transition states and, then, using this to (re)design enzymes to catalyze novel reactions remain the "holy grail" of mechanistic enzymology. The necessary foundation is the free-energy profile that specifies the energies of the bound substate, product, and intervening intermediates as well as the transition states by which they are interconverted. When this free-energy profile is compared to that for the uncatalyzed reaction, strategies for establishing and enhancing catalysis can be identified. This Perspective reminds readers that the first free-energy profile determined for an enzyme-catalyzed reaction, that for triosephosphate isomerase, was published in Biochemistry in 1976 by Jeremy R. Knowles, W. John Albery, and co-workers. They used the profile to propose three steps of increasing "subtlety" that can be influenced by evolutionary pressure to increase the flux through the reaction coordinate: (1) "uniform binding" of the substrate, product, and intermediates; (2) "differential binding" of complexes so that these are isoenergetic (to minimize the energy of the intervening transition states); and (3) "catalysis of an elementary step" in which the transition state for the kinetically significant chemical step is stabilized so that flux can be determined by the rate of substrate binding or product dissociation. These papers continue to guide mechanistic studies of enzyme-catalyzed reactions and provide principles for the (re)design of novel enzymes.

Discovery of new enzymatic functions and metabolic pathways using genomic enzymology web tools.

The continuing expansion of protein and genome sequence databases is an opportunity to identify novel enzymes with biotechnological applications. Whether applied to enzymology, chemical biology, systems biology, and microbiology, database mining must be 'user-friendly' so that experimentalists can devise focused strategies to discover the in vitro activities and in vivo functions of uncharacterized enzymes. We developed a suite of genomic enzymology tools (https://efi.igb.illinois.edu/) to (1) generate sequence similarity networks (SSNs) for exploration of sequence-function space in protein families (EFI-EST) and (2) provide genome context for members of protein families (EFI-GNT). Integrated analysis of this complementary information allows to generate testable hypotheses about new functions. After a brief overview of EFI-EST and EFI-GNT, we describe applications that illustrate their use.

Discovery of novel pathways for carbohydrate metabolism.

Closing the gap between the increasing availability of complete genome sequences and the discovery of novel enzymes in novel metabolic pathways is a significant challenge. Here, we review recent examples of assignment of in vitro enzymatic activities and in vivo metabolic functions to uncharacterized proteins, with a focus on enzymes and metabolic pathways involved in the catabolism and biosynthesis of monosaccharides and polysaccharides. The most effective approaches are based on analyses of sequence-function space in protein families that provide clues for the predictions of the functions of the uncharacterized enzymes. As summarized in this Opinion, this approach allows the discovery of the catabolism of new molecules, new pathways for common molecules, and new enzymatic chemistries.

Characterization of an l-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations.

l-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. Here we report that Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) uses l-ascorbate as sole carbon source via a novel catabolic pathway. RNaseq identified eight candidate catabolic genes, sequence similarity networks, and genome neighborhood networks guided predictions for function of the encoded proteins, and the predictions were confirmed by in vitro assays and in vivo growth phenotypes of gene deletion mutants. l-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b561 and gluconolactonase families, respectively, to form 2,3-diketo-l-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-l-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-l-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD+ catalytically. The product, l-lyxonate, is catabolized to α-ketoglutarate by a previously characterized pathway. The pathway is found in hundreds of bacteria, including the pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.

A bifunctional salvage pathway for two distinct S-adenosylmethionine by-products that is widespread in bacteria, including pathogenic Escherichia coli.

S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.

The gastrointestinal pathogen Campylobacter jejuni metabolizes sugars with potential help from commensal Bacteroides vulgatus.

Although the gastrointestinal pathogen Campylobacter jejuni was considered asaccharolytic, >50% of sequenced isolates possess an operon for L-fucose utilization. In C. jejuni NCTC11168, this pathway confers L-fucose chemotaxis and competitive colonization advantages in the piglet diarrhea model, but the catabolic steps remain unknown. Here we solved the putative dehydrogenase structure, resembling FabG of Burkholderia multivorans. The C. jejuni enzyme, FucX, reduces L-fucose and D-arabinose in vitro and both sugars are catabolized by fuc-operon encoded enzymes. This enzyme alone confers chemotaxis to both sugars in a non-carbohydrate-utilizing C. jejuni strain. Although C. jejuni lacks fucosidases, the organism exhibits enhanced growth in vitro when co-cultured with Bacteroides vulgatus, suggesting scavenging may occur. Yet, when excess amino acids are available, C. jejuni prefers them to carbohydrates, indicating a metabolic hierarchy exists. Overall this study increases understanding of nutrient metabolism by this pathogen, and identifies interactions with other gut microbes.

Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens.

Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ0 and preQ1 also occurs. With the exception of Escherichia coli YhhQ, shown to transport preQ0 and preQ1, the enzymes and transporters involved in Q salvage and recycling have not been well described. We discovered and characterized 2 Q salvage pathways present in many pathogenic and commensal bacteria. The first, found in the intracellular pathogen Chlamydia trachomatis, uses YhhQ and tRNA guanine transglycosylase (TGT) homologs that have changed substrate specificities to directly salvage q, mimicking the eukaryotic pathway. The second, found in bacteria from the gut flora such as Clostridioides difficile, salvages preQ1 from q through an unprecedented reaction catalyzed by a newly defined subgroup of the radical-SAM enzyme family. The source of q can be external through transport by members of the energy-coupling factor (ECF) family or internal through hydrolysis of Q by a dedicated nucleosidase. This work reinforces the concept that hosts and members of their associated microbiota compete for the salvage of Q precursors micronutrients.

The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways.

The assignment of functions to uncharacterized proteins discovered in genome projects requires easily accessible tools and computational resources for large-scale, user-friendly leveraging of the protein, genome, and metagenome databases by experimentalists. This article describes the web resource developed by the Enzyme Function Initiative (EFI; accessed at https://efi.igb.illinois.edu/ ) that provides "genomic enzymology" tools ("web tools") for (1) generating sequence similarity networks (SSNs) for protein families (EFI-EST); (2) analyzing and visualizing genome context of the proteins in clusters in SSNs (in genome neighborhood networks, GNNs, and genome neighborhood diagrams, GNDs) (EFI-GNT); and (3) prioritizing uncharacterized SSN clusters for functional assignment based on metagenome abundance (chemically guided functional profiling, CGFP) (EFI-CGFP). The SSNs generated by EFI-EST are used as the input for EFI-GNT and EFI-CGFP, enabling easy transfer of information among the tools. The networks are visualized and analyzed using Cytoscape, a widely used desktop application; GNDs and CGFP heatmaps summarizing metagenome abundance are viewed within the tools. We provide a detailed example of the integrated use of the tools with an analysis of glycyl radical enzyme superfamily (IPR004184) found in the human gut microbiome. This analysis demonstrates that (1) SwissProt annotations are not always correct, (2) large-scale genome context analyses allow the prediction of novel metabolic pathways, and (3) metagenome abundance can be used to identify/prioritize uncharacterized proteins for functional investigation.

Novel Metabolic Pathways and Regulons for Hexuronate Utilization in Proteobacteria.

We used comparative genomics to reconstruct d-galacturonic and d-glucuronic acid catabolic pathways and associated transcriptional regulons involving the tripartite ATP-independent periplasmic (TRAP) family transporters that bind hexuronates in proteobacteria. The reconstructed catabolic network involves novel transcription factors, catabolic enzymes, and transporters for utilization of both hexuronates and aldarates (d-glucarate and meso-galactarate). The reconstructed regulons for a novel GntR family transcription factor, GguR, include the majority of hexuronate/aldarate utilization genes in 47 species from the Burkholderiaceae, Comamonadaceae, Halomonadaceae, and Pseudomonadaceae families. GudR, GulR, and UdhR are additional local regulators of some hexuronate/aldarate utilization genes in some of the above-mentioned organisms. The predicted DNA binding motifs of GguR and GudR regulators from Ralstonia pickettii and Polaromonas were validated by in vitro binding assays. Genes from the GulR- and GguR-controlled loci were differentially expressed in R. pickettii grown on hexuronates and aldarates. By a combination of bioinformatics and experimental techniques we identified a novel variant of the oxidative pathway for hexuronate utilization, including two previously uncharacterized subfamilies of lactone hydrolases (UxuL and UxuF). The genomic context of respective genes and reconstruction of associated pathways suggest that both enzymes catalyze the conversion of d-galactaro- and d-glucaro-1,5-lactones to the ring-opened aldarates. The activities of the purified recombinant enzymes, UxuL and UxuF, from four proteobacterial species were directly confirmed and kinetically characterized. The inferred novel aldarate-specific transporter from the tripartite tricarboxylate transporter (TTT) family transporter TctC was confirmed to bind d-glucarate in vitro This study expands our knowledge of bacterial carbohydrate catabolic pathways by identifying novel families of catabolic enzymes, transcriptional regulators, and transporters.IMPORTANCE Hexuronate catabolic pathways and their transcriptional networks are highly variable among different bacteria. We identified novel transcriptional regulators that control the hexuronate and aldarate utilization genes in four families of proteobacteria. By regulon reconstruction and genome context analysis we identified several novel components of the common hexuronate/aldarate utilization pathways, including novel uptake transporters and catabolic enzymes. Two novel families of lactonases involved in the oxidative pathway of hexuronate catabolism were characterized. Novel transcriptional regulons were validated via in vitro binding assays and gene expression studies with Polaromonas and Ralstonia species. The reconstructed catabolic pathways are interconnected with each other metabolically and coregulated via the GguR regulons in proteobacteria.

Enzyme Architecture: Breaking Down the Catalytic Cage that Activates Orotidine 5'-Monophosphate Decarboxylase for Catalysis.

We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5'-monophosphate decarboxylase ( ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the -CH2OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter kcat/ Km for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on Δ G⧧ for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6-7.6) = 4.0 kcal/mol decrease in transition state stabilization from interactions with Q215, Y217, and R235. Mutant cycles show that ca. 2 kcal/mol of this 4 kcal/mol effect is from the direct interaction between the S154 and Q215 side chains and that ca. 2 kcal/mol is from a tightening in the stabilizing interactions of the Y217 and R235 side chains. The sum of the effects of individual A154S, A215Q, F217Y and A235R substitutions at the quadruple mutant of ScOMPDC to give the corresponding triple mutants, 5.6 kcal/mol, is much smaller than 16.0 kcal/mol, the sum of the effects of the related four substitutions in wild-type ScOMPDC to give the respective single mutants. The small effect of substitutions at the quadruple mutant is consistent with a large entropic cost to holding the flexible loops of ScOMPDC in the active closed conformation.

'Democratized' genomic enzymology web tools for functional assignment.

The protein databases contain an exponentially growing number of sequences as a result of the recent increase in ease and decrease in cost of genome sequencing. The rate of data accumulation far exceeds the rate of functional studies, producing an increase in genomic 'dark matter', sequences for which no precise and validated function is defined. Publicly accessible, that is 'democratized,' genomic enzymology web tools are essential to leverage the protein and genome databases for discovery of the in vitro activities and in vivo functions of novel enzymes and proteins belonging to the dark matter. In this review, we discuss the use of web tools that have proven successful for functional assignment. We also describe a mechanism for ensuring the capture of published functional data so that the quality of both curated and automated annotations transfer can be improved.

Functional assignment of multiple catabolic pathways for D-apiose.

Colocation of the genes encoding ABC, TRAP, and TCT transport systems and catabolic pathways for the transported ligand provides a strategy for discovering novel microbial enzymes and pathways. We screened solute-binding proteins (SBPs) for ABC transport systems and identified three that bind D-apiose, a branched pentose in the cell walls of higher plants. Guided by sequence similarity networks (SSNs) and genome neighborhood networks (GNNs), the identities of the SBPs enabled the discovery of four catabolic pathways for D-apiose with eleven previously unknown reactions. The new enzymes include D-apionate oxidoisomerase, which catalyzes hydroxymethyl group migration, as well as 3-oxo-isoapionate-4-phosphate decarboxylase and 3-oxo-isoapionate-4-phosphate transcarboxylase/hydrolase, which are RuBisCO-like proteins (RLPs). The web tools for generating SSNs and GNNs are publicly accessible ( http://efi.igb.illinois.edu/efi-est/ ), so similar 'genomic enzymology' strategies for discovering novel pathways can be used by the community.

Prediction of enzymatic pathways by integrative pathway mapping.

The functions of most proteins are yet to be determined. The function of an enzyme is often defined by its interacting partners, including its substrate and product, and its role in larger metabolic networks. Here, we describe a computational method that predicts the functions of orphan enzymes by organizing them into a linear metabolic pathway. Given candidate enzyme and metabolite pathway members, this aim is achieved by finding those pathways that satisfy structural and network restraints implied by varied input information, including that from virtual screening, chemoinformatics, genomic context analysis, and ligand -binding experiments. We demonstrate this integrative pathway mapping method by predicting the L-gulonate catabolic pathway in Haemophilus influenzae Rd KW20. The prediction was subsequently validated experimentally by enzymology, crystallography, and metabolomics. Integrative pathway mapping by satisfaction of structural and network restraints is extensible to molecular networks in general and thus formally bridges the gap between structural biology and systems biology.