Crystal structures of Moorella thermoacetica cyanuric acid hydrolase reveal conformational flexibility and asymmetry important for catalysis.

An ancient enzyme family responsible for the catabolism of the prebiotic chemical cyanuric acid (1,3,5-triazine-2,4,6-triol) was recently discovered and is undergoing proliferation in the modern world due to industrial synthesis and dissemination of 1,3,5-triazine compounds. Cyanuric acid has a highly stabilized ring system such that bacteria require a unique enzyme with a novel fold and subtle active site construction to open the ring. Each cyanuric acid hydrolase monomer consists of three isostructural domains that coordinate and activate the three-fold symmetric substrate cyanuric acid for ring opening. We have now solved a series of X-ray structures of an engineered, thermostable cyanuric acid ring-opening enzyme at 1.51 ~ 2.25 Å resolution, including various complexes with the substrate, a tight-binding inhibitor, or an analog of the reaction intermediate. These structures reveal asymmetric interactions between the enzyme and bound ligands, a metal ion binding coupled to conformational changes and substrate binding important for enzyme stability, and distinct roles of the isostructural domains of the enzyme. The multiple conformations of the enzyme observed across a series of structures and corroborating biochemical data suggest importance of the structural dynamics in facilitating the substrate entry and the ring-opening reaction, catalyzed by a conserved Ser-Lys dyad.

Ancient Evolution and Recent Evolution Converge for the Biodegradation of Cyanuric Acid and Related Triazines.

Cyanuric acid was likely present on prebiotic Earth, may have been a component of early genetic materials, and is synthesized industrially today on a scale of more than one hundred million pounds per year in the United States. In light of this, it is not surprising that some bacteria and fungi have a metabolic pathway that sequentially hydrolyzes cyanuric acid and its metabolites to release the nitrogen atoms as ammonia to support growth. The initial reaction that opens the s-triazine ring is catalyzed by the unusual enzyme cyanuric acid hydrolase. This enzyme is in a rare protein family that consists of only cyanuric acid hydrolase (CAH) and barbiturase, with barbiturase participating in pyrimidine catabolism by some actinobacterial species. The X-ray structures of two cyanuric acid hydrolase proteins show that this family has a unique protein fold. Phylogenetic, bioinformatic, enzymological, and genetic studies are consistent with the idea that CAH has an ancient protein fold that was rare in microbial populations but is currently becoming more widespread in microbial populations in the wake of anthropogenic synthesis of cyanuric acid and other s-triazine compounds that are metabolized via a cyanuric acid intermediate. The need for the removal of cyanuric acid from swimming pools and spas, where it is used as a disinfectant stabilizer, can potentially be met using an enzyme filtration system. A stable thermophilic cyanuric acid hydrolase from Moorella thermoacetica is being tested for this purpose.

Characteristic isotope fractionation patterns in s-triazine degradation have their origin in multiple protonation options in the s-triazine hydrolase TrzN.

s-Triazine herbicides (atrazine, ametryn) are groundwater contaminants which may undergo microbial hydrolysis. Previously, inverse nitrogen isotope effects in atrazine degradation by Arthrobacter aurescens TC1 (i) delivered highly characteristic (13C/12C, 15N/14N) fractionation trends for pathway identification and (ii) suggested that the s-triazine ring nitrogen was protonated in the enzyme s-triazine hydrolase (TrzN) where (iii) TrzN crystal structure and mutagenesis indicated H+-transfer from the residue E241. This study tested the general validity of these conclusions for atrazine and ametryn with purified TrzN and a TrzN-E241Q site-directed mutant. TrzN-E241Q lacked activity with ametryn; otherwise, degradation consistently showed normal carbon isotope effects (εcarbon=-5.0‰±0.2‰ (atrazine/TrzN), εcarbon=-4.2‰±0.5‰ (atrazine/TrzN-E241Q), εcarbon=-2.4‰±0.3‰ (ametryn/TrzN)) and inverse nitrogen isotope effects (εnitrogen=2.5‰±0.1‰ (atrazine/TrzN), εnitrogen=2.1‰±0.3‰ (atrazine/TrzN-E241Q), εnitrogen=3.6‰±0.4‰ (ametryn/TrzN)). Surprisingly, TrzN-E241Q therefore still activated substrates through protonation implicating another proton donor besides E241. Sulfur isotope effects were larger in enzymatic (εsulfur=-14.7‰±1.0‰, ametryn/TrzN) than in acidic ametryn hydrolysis (εsulfur=-0.2‰±0.0‰, pH 1.75), indicating rate-determining C-S bond cleavage in TrzN. Our results highlight a robust inverse 15N/14N fractionation pattern for identifying microbial s-triazine hydrolysis in the environment caused by multiple protonation options in TrzN.

Cyanuric acid hydrolase from Azorhizobium caulinodans ORS 571: crystal structure and insights into a new class of Ser-Lys dyad proteins.

Cyanuric acid hydrolase (CAH) catalyzes the hydrolytic ring-opening of cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine), an intermediate in s-triazine bacterial degradation and a by-product from disinfection with trichloroisocyanuric acid. In the present study, an X-ray crystal structure of the CAH-barbituric acid inhibitor complex from Azorhizobium caulinodans ORS 571 has been determined at 2.7 Å resolution. The CAH protein fold consists of three structurally homologous domains forming a ß-barrel-like structure with external α-helices that result in a three-fold symmetry, a dominant feature of the structure and active site that mirrors the three-fold symmetrical shape of the substrate cyanuric acid. The active site structure of CAH is similar to that of the recently determined AtzD with three pairs of active site Ser-Lys dyads. In order to determine the role of each Ser-Lys dyad in catalysis, a mutational study using a highly sensitive, enzyme-coupled assay was conducted. The 109-fold loss of activity by the S226A mutant was at least ten times lower than that of the S79A and S333A mutants. In addition, bioinformatics analysis revealed the Ser226/Lys156 dyad as the only absolutely conserved dyad in the CAH/barbiturase family. These data suggest that Lys156 activates the Ser226 nucleophile which can then attack the substrate carbonyl. Our combination of structural, mutational, and bioinformatics analyses differentiates this study and provides experimental data for mechanistic insights into this unique protein family.

Caffeine junkie: an unprecedented glutathione S-transferase-dependent oxygenase required for caffeine degradation by Pseudomonas putida CBB5.

Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N1- and N3-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N7-demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners.

Defining sequence space and reaction products within the cyanuric acid hydrolase (AtzD)/barbiturase protein family.

Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.

X-ray structure and mutational analysis of the atrazine Chlorohydrolase TrzN.

Atrazine chlorohydrolase, TrzN (triazine hydrolase or atrazine chlorohydrolase 2), initiates bacterial metabolism of the herbicide atrazine by hydrolytic displacement of a chlorine substituent from the s-triazine ring. The present study describes crystal structures and reactivity of wild-type and active site mutant TrzN enzymes. The homodimer native enzyme structure, solved to 1.40 Å resolution, is a (ßα)(8) barrel, characteristic of members of the amidohydrolase superfamily. TrzN uniquely positions threonine 325 in place of a conserved aspartate that ligates the metal in most mononuclear amidohydrolases superfamily members. The threonine side chain oxygen atom is 3.3 Å from the zinc atom and 2.6 Å from the oxygen atom of zinc-coordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in k(cat)/K(m), largely due to k(cat), whereas the T325D and T325E mutants had immeasurable activity. The structure and kinetics of TrzN are reminiscent of carbonic anhydrase, which uses a threonine to assist in positioning water for reaction with carbon dioxide. An isosteric substitution in the active site glutamate, E241Q, showed a large diminution in activity with ametryn, no detectable activity with atratone, and a 10-fold decrease with atrazine, when compared with wild-type TrzN. Activity with the E241Q mutant was nearly constant from pH 6.0 to 10.0, consistent with the loss of a proton-donating group. Structures for TrzN-E241Q were solved with bound ametryn and atratone to 1.93 and 1.64 Å resolution, respectively. Both structure and kinetic determinations suggest that the Glu(241) side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.

Widespread head-to-head hydrocarbon biosynthesis in bacteria and role of OleA.

Previous studies identified the oleABCD genes involved in head-to-head olefinic hydrocarbon biosynthesis. The present study more fully defined the OleABCD protein families within the thiolase, alpha/beta-hydrolase, AMP-dependent ligase/synthase, and short-chain dehydrogenase superfamilies, respectively. Only 0.1 to 1% of each superfamily represents likely Ole proteins. Sequence analysis based on structural alignments and gene context was used to identify highly likely ole genes. Selected microorganisms from the phyla Verucomicrobia, Planctomyces, Chloroflexi, Proteobacteria, and Actinobacteria were tested experimentally and shown to produce long-chain olefinic hydrocarbons. However, different species from the same genera sometimes lack the ole genes and fail to produce olefinic hydrocarbons. Overall, only 1.9% of 3,558 genomes analyzed showed clear evidence for containing ole genes. The type of olefins produced by different bacteria differed greatly with respect to the number of carbon-carbon double bonds. The greatest number of organisms surveyed biosynthesized a single long-chain olefin, 3,6,9,12,15,19,22,25,28-hentriacontanonaene, that contains nine double bonds. Xanthomonas campestris produced the greatest number of distinct olefin products, 15 compounds ranging in length from C(28) to C(31) and containing one to three double bonds. The type of long-chain product formed was shown to be dependent on the oleA gene in experiments with Shewanella oneidensis MR-1 ole gene deletion mutants containing native or heterologous oleA genes expressed in trans. A strain deleted in oleABCD and containing oleA in trans produced only ketones. Based on these observations, it was proposed that OleA catalyzes a nondecarboxylative thiolytic condensation of fatty acyl chains to generate a beta-ketoacyl intermediate that can decarboxylate spontaneously to generate ketones.

Structure, function, and insights into the biosynthesis of a head-to-head hydrocarbon in Shewanella oneidensis strain MR-1.

A polyolefinic hydrocarbon was found in nonpolar extracts of Shewanella oneidensis MR-1 and identified as 3,6,9,12,15,19,22,25,28-hentriacontanonaene (compound I) by mass spectrometry, chemical modification, and nuclear magnetic resonance spectroscopy. Compound I was shown to be the product of a head-to-head fatty acid condensation biosynthetic pathway dependent on genes denoted as ole (for olefin biosynthesis). Four ole genes were present in S. oneidensis MR-1. Deletion of the entire oleABCD gene cluster led to the complete absence of nonpolar extractable products. Deletion of the oleC gene alone generated a strain that lacked compound I but produced a structurally analogous ketone. Complementation of the oleC gene eliminated formation of the ketone and restored the biosynthesis of compound I. A recombinant S. oneidensis strain containing oleA from Stenotrophomonas maltophilia strain R551-3 produced at least 17 related long-chain compounds in addition to compound I, 13 of which were identified as ketones. A potential role for OleA in head-to-head condensation was proposed. It was further proposed that long-chain polyunsaturated compounds aid in adapting cells to a rapid drop in temperature, based on three observations. In S. oneidensis wild-type cells, the cellular concentration of polyunsaturated compounds increased significantly with decreasing growth temperature. Second, the oleABCD deletion strain showed a significantly longer lag phase than the wild-type strain when shifted to a lower temperature. Lastly, compound I has been identified in a significant number of bacteria isolated from cold environments.

Bacterial ammeline metabolism via guanine deaminase.

Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.

Thermostable cyanuric acid hydrolase from Moorella thermoacetica ATCC 39073.

Cyanuric acid, a metabolic intermediate in the degradation of many s-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile Moorella thermoacetica ATCC 39073 was cloned, expressed in Escherichia coli, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and N-methylisocyanuric acid. The M. thermoacetica enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from M. thermoacetica is the most promising enzyme to use for cyanuric acid remediation applications.

Investigative mining of sequence data for novel enzymes: a case study with nitrilases.

Mining sequence data is increasingly important for biocatalysis research. However, when relying on sequence data alone, prediction of the reaction catalyzed by a specific protein sequence is often elusive, and substrate specificity is far from trivial. The present study demonstrated an approach of combining sequence data and structures from distant homologs to target identification of new nitrilases that specifically utilize hindered nitrile substrates like mandelonitrile. A total of 212 non-identical target nitrilases were identified from GenBank. Evolutionary trace and sequence clustering methods were used combinatorily to identify a set of nitrilases with presumably distinct substrate specificities. Selected encoding genes were cloned into Escherichia coli. Recombinant E. coli expressing NitA (gi91784632) from Burkholderia xenovorans LB400 was capable of growth on glutaronitrile or adiponitrile as the sole nitrogen source. Purified NitA exhibited highest activity with mandelonitrile, showing a catalytic efficiency (k(cat)/K(m)) of 3.6 x 10(4)M(-1)s(-1). A second nitrilase predicted from our studies from Bradyrhizobium zaponicum USDA 110 (gi27381513) was likewise shown to prefer mandelonitrile [Zhu, D., Mukherjee, C., Biehl, E.R., Hua, L., 2007. Discovery of a mandelonitrile hydrolase from Bradyrhizobium japonicum USDA110 by rational genome mining. J. Biotechnol. 129 (4), 645-650]. Thus, predictions from sequence analysis and distant superfamily structures yielded enzyme activities with high selectivity for mandelonitrile. These data suggest that similar data mining techniques can be used to identify other substrate-specific enzymes from published, unannotated sequences.

Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1.

Vibrio furnissii M1 was recently reported to biosynthesize n-alkanes when grown on biopolymers, sugars, or organic acids (M. O. Park, J. Bacteriol. 187:1426-1429, 2005). In the present study, V. furnissii M1 was subjected to genomic analysis and studied biochemically. The sequence of the 16S rRNA gene and repetitive PCR showed that V. furnissii M1 was not identical to other V. furnissii strains tested, but the level of relatedness was consistent with its assignment as a V. furnissii strain. Pulsed-field gel electrophoresis showed chromosomal bands at approximately 3.2 and 1.8 Mb, similar to other Vibrio strains. Complete genomic DNA from V. furnissii M1 was sequenced with 21-fold coverage. Alkane biosynthetic and degradation genes could not be identified. Moreover, V. furnissii M1 did not produce demonstrable levels of n-alkanes in vivo or in vitro. In vivo experiments were conducted by growing V. furnissii M1 under different conditions, extracting with solvent, and analyzing extracts by gas chromatography-mass spectrometry. A highly sensitive assay was used for in vitro experiments with cell extracts and [(14)C]hexadecanol. The data are consistent with the present strain being a V. furnissii with properties similar to those previously described but lacking the alkane-producing phenotype. V. furnissii ATCC 35016, also reported to biosynthesize alkanes, was found in the present study not to produce alkanes.

Hydroxyatrazine N-ethylaminohydrolase (AtzB): an amidohydrolase superfamily enzyme catalyzing deamination and dechlorination.

Hydroxyatrazine [2-(N-ethylamino)-4-hydroxy-6-(N-isopropylamino)-1,3,5-triazine] N-ethylaminohydrolase (AtzB) is the sole enzyme known to catalyze the hydrolytic conversion of hydroxyatrazine to N-isopropylammelide. AtzB, therefore, serves as the point of intersection of multiple s-triazine biodegradative pathways and is completely essential for microbial growth on s-triazine herbicides. Here, atzB was cloned from Pseudomonas sp. strain ADP and its product was purified to homogeneity and characterized. AtzB was found to be dimeric, with subunit and holoenzyme molecular masses of 52 kDa and 105 kDa, respectively. The k(cat) and K(m) of AtzB with hydroxyatrazine as a substrate were 3 s(-1) and 20 microM, respectively. Purified AtzB had a 1:1 zinc-to-subunit stoichiometry. Sequence analysis revealed that AtzB contained the conserved mononuclear amidohydrolase superfamily active-site residues His74, His76, His245, Glu248, His280, and Asp331. An intensive in vitro investigation into the substrate specificity of AtzB revealed that 20 of the 51 compounds tested were substrates for AtzB; this allowed for the identification of specific substrate structural features required for catalysis. Substrates required a monohydroxylated s-triazine ring with a minimum of one primary or secondary amine substituent and either a chloride or amine leaving group. AtzB catalyzed both deamination and dechlorination reactions with rates within a range of one order of magnitude. This differs from AtzA and TrzN, which do not catalyze deamination reactions, and AtzC, which is not known to catalyze dechlorination reactions.

A gold standard set of mechanistically diverse enzyme superfamilies.

Superfamily and family analyses provide an effective tool for the functional classification of proteins, but must be automated for use on large datasets. We describe a 'gold standard' set of enzyme superfamilies, clustered according to specific sequence, structure, and functional criteria, for use in the validation of family and superfamily clustering methods. The gold standard set represents four fold classes and differing clustering difficulties, and includes five superfamilies, 91 families, 4,887 sequences and 282 structures.

Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme.

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.

Enzymatic degradation of chlorodiamino-s-triazine.

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by N-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.