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.

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.

Discovery of new enzymes and metabolic pathways by using structure and genome context.

Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns. We and others are developing computation-guided strategies for functional discovery with 'metabolite docking' to experimentally derived or homology-based three-dimensional structures. Bacterial metabolic pathways often are encoded by 'genome neighbourhoods' (gene clusters and/or operons), which can provide important clues for functional assignment. We recently demonstrated the synergy of docking and pathway context by 'predicting' the intermediates in the glycolytic pathway in Escherichia coli. Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB 2PMQ), 2-epimerization of trans-4-hydroxy-L-proline betaine (tHyp-B) and cis-4-hydroxy-D-proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.

A novel bifunctional transcriptional regulator of riboflavin metabolism in Archaea.

Riboflavin (vitamin B2) is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide, which are essential coenzymes in all free-living organisms. Riboflavin biosynthesis in many Bacteria but not in Archaea is controlled by FMN-responsive riboswitches. We identified a novel bifunctional riboflavin kinase/regulator (RbkR), which controls riboflavin biosynthesis and transport genes in major lineages of Crenarchaeota, Euryarchaeota and Thaumarchaeota. RbkR proteins are composed of the riboflavin kinase domain and a DNA-binding winged helix-turn-helix-like domain. Using comparative genomics, we predicted RbkR operator sites and reconstructed RbkR regulons in 94 archaeal genomes. While the identified RbkR operators showed significant variability between archaeal lineages, the conserved core of RbkR regulons includes riboflavin biosynthesis genes, known/predicted vitamin uptake transporters and the rbkR gene. The DNA motifs and CTP-dependent riboflavin kinase activity of two RbkR proteins were experimentally validated in vitro. The DNA binding activity of RbkR was stimulated by CTP and suppressed by FMN, a product of riboflavin kinase. The crystallographic structure of RbkR from Thermoplasma acidophilum was determined in complex with CTP and its DNA operator revealing key residues for operator and ligand recognition. Overall, this study contributes to our understanding of metabolic and regulatory networks for vitamin homeostasis in Archaea.

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars.

Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for d-threonate, l-threonate, and d-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including d-erythronate and l-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-l-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of d-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of d-erythronate and l-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2 The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses.

Large-scale determination of sequence, structure, and function relationships in cytosolic glutathione transferases across the biosphere.

The cytosolic glutathione transferase (cytGST) superfamily comprises more than 13,000 nonredundant sequences found throughout the biosphere. Their key roles in metabolism and defense against oxidative damage have led to thousands of studies over several decades. Despite this attention, little is known about the physiological reactions they catalyze and most of the substrates used to assay cytGSTs are synthetic compounds. A deeper understanding of relationships across the superfamily could provide new clues about their functions. To establish a foundation for expanded classification of cytGSTs, we generated similarity-based subgroupings for the entire superfamily. Using the resulting sequence similarity networks, we chose targets that broadly covered unknown functions and report here experimental results confirming GST-like activity for 82 of them, along with 37 new 3D structures determined for 27 targets. These new data, along with experimentally known GST reactions and structures reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and reveal that the great majority of cytGSTs have not been experimentally characterized or annotated by canonical class. A mapping of taxonomic classes across the superfamily indicates that many taxa are represented in each subgroup and highlights challenges for classification of superfamily sequences into functionally relevant classes. Experimental determination of disulfide bond reductase activity in many diverse subgroups illustrate a theme common for many reaction types. Finally, sequence comparison between an enzyme that catalyzes a reductive dechlorination reaction relevant to bioremediation efforts with some of its closest homologs reveals differences among them likely to be associated with evolution of this unusual reaction. Interactive versions of the networks, associated with functional and other types of information, can be downloaded from the Structure-Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu).

ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58.

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool.

Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute binding proteins and unbiased panning of metabolomes.

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.

A unique cis-3-hydroxy-l-proline dehydratase in the enolase superfamily.

The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the enolase superfamily (UniProt ID A0NXQ8 ). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7 ; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9 ; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to Δ(1)-pyrroline-2-carboxylate (ß-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources.

A General Strategy for the Discovery of Metabolic Pathways: d-Threitol, l-Threitol, and Erythritol Utilization in Mycobacterium smegmatis.

We describe a general integrated bioinformatic and experimental strategy to discover the in vitro enzymatic activities and in vivo functions (metabolic pathways) of uncharacterized enzymes discovered in microbial genome projects using the ligand specificities of the solute binding proteins (SBPs) for ABC transporters. Using differential scanning fluorimetry, we determined that the SBP for an ABC transporter encoded by the genome of Mycobacterium smegmatis is stabilized by d-threitol. Using sequence similarity networks and genome neighborhood networks to guide selection of target proteins for pathway enzymes, we applied both in vitro and in vivo experimental approaches to discover novel pathways for catabolism of d-threitol, l-threitol, and erythritol.

Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily.

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion.

Galactaro δ-lactone isomerase: lactone isomerization by a member of the amidohydrolase superfamily.

Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of d-galacturonate to D-galactaro-1,5-lactone. We have identified a novel enzyme, D-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of D-galactaro-1,5-lactone to D-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase.

Evolution of enzymatic activities in the enolase superfamily: galactarate dehydratase III from Agrobacterium tumefaciens C58.

The genome of Agrobacterium tumefaciens C58 encodes 12 members of the enolase superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars for high-throughput screening, one protein (UniProt entry A9CG74; locus tag Atu4196) showed activity with both m-galactarate and d-galacturonate. Two families of galactarate dehydratases had been discovered previously in the ENS, GalrD/TalrD [Yew, W. S., et al. (2007) Biochemistry 46, 9564-9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry 48, 11546-11558]; these have different active site acid/base catalysis and have no activity with d-galacturonate. A9CG74 dehydrates m-galactarate to form 2-keto-3-deoxy-galactarate but does not dehydrate d-galacturonate as expected. Instead, when A9CG74 is incubated with d-galacturonate, 3-deoxy-d-xylo-hexarate or 3-deoxy-d-lyxo-hexarate is formed. In this reaction, instead of abstracting the C5 proton α to the carboxylate group, the expected reaction for a member of the ENS, the enzyme apparently abstracts the proton α to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to the benzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarate as a carbon source under the conditions tested in this study, although it does utilize d-galacturonate, which is a likely precursor to m-galactarate. The gene encoding A9CG74 and several genome proximal genes were upregulated with d-galacturonate as the carbon source. One of these, a member of the dihydrodipicolinate synthase superfamily, catalyzes the dehydration and subsequent decarboxylation of 2-keto-3-deoxy-d-galactarate to α-ketoglutarate semialdehyde, thereby providing a pathway for the conversion of m-galactarate to α-ketoglutarate semialdehyde.

Prospecting for unannotated enzymes: discovery of a 3',5'-nucleotide bisphosphate phosphatase within the amidohydrolase superfamily.

In bacteria, 3',5'-adenosine bisphosphate (pAp) is generated from 3'-phosphoadenosine 5'-phosphosulfate in the sulfate assimilation pathway, and from coenzyme A by the transfer of the phosphopantetheine group to the acyl-carrier protein. pAp is subsequently hydrolyzed to 5'-AMP and orthophosphate, and this reaction has been shown to be important for superoxide stress tolerance. Herein, we report the discovery of the first instance of an enzyme from the amidohydrolase superfamily that is capable of hydrolyzing pAp. Crystal structures of Cv1693 from Chromobacterium violaceum have been determined to a resolution of 1.9 Å with AMP and orthophosphate bound in the active site. The enzyme has a trinuclear metal center in the active site with three Mn(2+) ions. This enzyme (Cv1693) belongs to the Cluster of Orthologous Groups cog0613 from the polymerase and histidinol phosphatase family of enzymes. The values of kcat and kcat/Km for the hydrolysis of pAp are 22 s(-1) and 1.4 × 10(6) M(-1) s(-1), respectively. The enzyme is promiscuous and is able to hydrolyze other 3',5'-bisphosphonucleotides (pGp, pCp, pUp, and pIp) and 2'-deoxynucleotides with comparable catalytic efficiency. The enzyme is capable of hydrolyzing short oligonucleotides (pdA)5, albeit at rates much lower than that of pAp. Enzymes from two other enzyme families have previously been found to hydrolyze pAp at physiologically significant rates. These enzymes include CysQ from Escherichia coli (cog1218) and YtqI/NrnA from Bacillus subtilis (cog0618). Identification of the functional homologues to the experimentally verified pAp phosphatases from cog0613, cog1218, and cog0618 suggests that there is relatively little overlap of enzymes with this function in sequenced bacterial genomes.

Discovery of function in the enolase superfamily: D-mannonate and d-gluconate dehydratases in the D-mannonate dehydratase subgroup.

The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated ( Schnoes et al. PLoS Comput. Biol. 2009 , 5 ( 12 ), e1000605 ). This manuscript describes a study of the D-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of D-mannonate to 2-keto-3-deoxy-D-mannonate (equivalently, 2-keto-3-deoxy-D-gluconate)]. In this study, 42 additional members were characterized to sample sequence-function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 10(3) to 10(4) M(-1) s(-1)) for dehydration of D-mannonate, (2) low efficiency (kcat/KM = 10(1) to 10(2) M(-1) s(-1)) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes D-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004 , 576 , 133 - 136 ) (Ahmed et al. Biochem. J. 2005 , 390 , 529 - 540 ). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs.

Discovery of an L-fucono-1,5-lactonase from cog3618 of the amidohydrolase superfamily.

A member of the amidohydrolase superfamily, BmulJ_04915 from Burkholderia multivorans, of unknown function was determined to hydrolyze a series of sugar lactones: L-fucono-1,4-lactone, D-arabino-1,4-lactone, L-xylono-1,4-lactone, D-lyxono-1,4-lactone, and L-galactono-1,4-lactone. The highest activity was shown for L-fucono-1,4-lactone with a k(cat) value of 140 s(-1) and a k(cat)/K(m) value of 1.0 × 10(5) M(-1) s(-1) at pH 8.3. The enzymatic product of an adjacent L-fucose dehydrogenase, BmulJ_04919, was shown to be L-fucono-1,5-lactone via nuclear magnetic resonance spectroscopy. L-Fucono-1,5-lactone is unstable and rapidly converts nonenzymatically to L-fucono-1,4-lactone. Because of the chemical instability of L-fucono-1,5-lactone, 4-deoxy-L-fucono-1,5-lactone was enzymatically synthesized from 4-deoxy-L-fucose using L-fucose dehydrogenase. BmulJ_04915 hydrolyzed 4-deoxy-L-fucono-1,5-lactone with a k(cat) value of 990 s(-1) and a k(cat)/K(m) value of 8.0 × 10(6) M(-1) s(-1) at pH 7.1. The protein does not require divalent cations in the active site for catalytic activity. BmulJ_04915 is the second enzyme from cog3618 of the amidohydrolase superfamily that does not require a divalent metal for catalytic activity. BmulJ_04915 is the first enzyme that has been shown to catalyze the hydrolysis of either L-fucono-1,4-lactone or L-fucono-1,5-lactone. The structures of the fuconolactonase and the fucose dehydrogenase were determined by X-ray diffraction methods.

Structure-guided discovery of new deaminase enzymes.

A substantial challenge for genomic enzymology is the reliable annotation for proteins of unknown function. Described here is an interrogation of uncharacterized enzymes from the amidohydrolase superfamily using a structure-guided approach that integrates bioinformatics, computational biology, and molecular enzymology. Previously, Tm0936 from Thermotoga maritima was shown to catalyze the deamination of S-adenosylhomocysteine (SAH) to S-inosylhomocysteine (SIH). Homologues of Tm0936 homologues were identified, and substrate profiles were proposed by docking metabolites to modeled enzyme structures. These enzymes were predicted to deaminate analogues of adenosine including SAH, 5'-methylthioadenosine (MTA), adenosine (Ado), and 5'-deoxyadenosine (5'-dAdo). Fifteen of these proteins were purified to homogeneity, and the three-dimensional structures of three proteins were determined by X-ray diffraction methods. Enzyme assays supported the structure-based predictions and identified subgroups of enzymes with the capacity to deaminate various combinations of the adenosine analogues, including the first enzyme (Dvu1825) capable of deaminating 5'-dAdo. One subgroup of proteins, exemplified by Moth1224 from Moorella thermoacetica, deaminates guanine to xanthine, and another subgroup, exemplified by Avi5431 from Agrobacterium vitis S4, deaminates two oxidatively damaged forms of adenine: 2-oxoadenine and 8-oxoadenine. The sequence and structural basis of the observed substrate specificities were proposed, and the substrate profiles for 834 protein sequences were provisionally annotated. The results highlight the power of a multidisciplinary approach for annotating enzymes of unknown function.

Structure of QnrB1, a plasmid-mediated fluoroquinolone resistance factor.

QnrB1 is a plasmid-encoded pentapeptide repeat protein (PRP) that confers a moderate degree of resistance to fluoroquinolones. Its gene was cloned into an expression vector with an N-terminal polyhistidine tag, and the protein was purified by nickel affinity chromatography. The structure of QnrB1 was determined by a combination of trypsinolysis, surface mutagenesis, and single anomalous dispersion phasing. QnrB1 folds as a right-handed quadrilateral ß-helix with a highly asymmetric dimeric structure typical of PRP-topoisomerase poison resistance factors. The threading of pentapeptides into the ß-helical fold is interrupted by two noncanonical PRP sequences that produce outward projecting loops that interrupt the regularity of the PRP surface. Deletion of the larger upper loop eliminated the protective effect of QnrB1 on DNA gyrase toward inhibition by quinolones, whereas deletion of the smaller lower loop drastically reduced the protective effect. These loops are conserved among all plasmid-based Qnr variants (QnrA, QnrC, QnrD, and QnrS) and some chromosomally encoded Qnr varieties. A mechanism in which PRP-topoisomerase poison resistance factors bind to and disrupt the quinolone-DNA-gyrase interaction is proposed.

The structure and mechanism of the Mycobacterium tuberculosis cyclodityrosine synthetase.

The Mycobacterium tuberculosis enzyme Rv2275 catalyzes the formation of cyclo(L-Tyr-L-Tyr) using two molecules of Tyr-tRNA(Tyr) as substrates. The three-dimensional (3D) structure of Rv2275 was determined to 2.0-Å resolution, revealing that Rv2275 is structurally related to the class Ic aminoacyl-tRNA synthetase family of enzymes. Mutagenesis and radioactive labeling suggests a covalent intermediate in which L-tyrosine is transferred from Tyr-tRNA(Tyr) to an active site serine (Ser88) by transesterification with Glu233 serving as a critical base, catalyzing dipeptide bond formation.