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Protein Family to the Fungal Kingdom Expanding the Cyanuric Acid

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Protein Family to the Fungal Kingdom Expanding the Cyanuric Acid Downloaded from http://jb.asm.org/ on November 6, 2013 by guest more» 2013, 195(23):5233. DOI: 10.1128/JB.00965-13. http://journals.asm.org/site/subscriptions/ http://jb.asm.org/content/195/23/5233#ref-list-1 http://journals.asm.org/site/misc/reprints.xhtml Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), This article cites 39 articles, 20 of which can be accessed free at: Updated information and services can be found at: http://jb.asm.org/content/195/23/5233 These include: Supplemental material Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom Anthony G. Dodge, Chelsea S. Preiner and Lawrence P. Wackett J. Bacteriol. Published Ahead of Print 13 September 2013. REFERENCES CONTENT ALERTS To subscribe to to another ASM Journal go to: SUPPLEMENTAL MATERIAL Information about commercial reprint orders: Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom Anthony G. Dodge,a Chelsea S. Preiner,a Lawrence P. Wacketta,b BioTechnology Institute,a and Department of Biochemistry, Molecular Biology, and Biophysics,b University of Minnesota, St. Paul, Minnesota, USA The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the pres- ent study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two ex- ons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decar- boxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. yanuric acid was first synthesized in 1829 by Wöhler (1) and is (18). The barbiturase reaction is part of the oxidative pyrimidine Ccurrently an important commercial compound and interme- salvage pathway and has thus far been reported to occur only in diate. As examples of the latter, it is used to prepare cross-linked the Actinobacteria (18, 19). The sequence divergence among char- polymers and chlorinated cyanuric acid for disinfection. Cyanuric acterized cyanuric acid hydrolases and barbiturases suggests an acid alone is only mildly toxic, but coingestion with melamine is ancient origin for these enzymes, making it surprising that the extremely hazardous. Recently, melamine containing significant range of reactions catalyzed by this protein family is relatively levels of cyanuric acid was used to adulterate pet food, and insol- narrow (11, 14). Cyanuric acid degradation by several fungi has uble melamine-cyanuric acid complexes (2, 3) were identified as been described (20–23), but the genetic and enzymatic basis for the etiological agents of kidney failure in animals that ingested degradation was not elucidated in those previous studies. food made with the tainted ingredients (4). In the present study, we isolated a filamentous fungus by en- Cyanuric acid is also formed as a metabolic intermediate dur- richment culturing that grew by using cyanuric acid as the sole ing the microbial metabolism of s-triazine compounds such as nitrogen source. A cyanuric acid hydrolase gene homolog was melamine (5) and the herbicide atrazine (6). This metabolic capa- identified in the strain by reverse genetics, and the translated se- bility is found within only a very limited number of bacteria. These quence, minus the intron regions, was observed to be homologous bacteria transform cyanuric acid to 3 mol each of ammonia and to known bacterial cyanuric acid hydrolases. A bacterially express- carbon dioxide (7) in a three-enzyme hydrolytic pathway. The ible gene was designed, the encoded polypeptide was purified, and pathway begins with cleavage of the s-triazine ring by the enzyme the protein was shown to catalyze cyanuric acid hydrolysis effi- cyanuric acid hydrolase, which produces biuret (8–11). Subse- ciently and to not act on many other analogous compounds. The quently, biuret undergoes enzymatic hydrolysis with the release of hydrolysis reaction produced carboxybiuret that decomposed ammonia by biuret hydrolase (9, 12), and the other product, al- spontaneously to biuret, as shown by 13C nuclear magnetic reso- lophanate, is hydrolyzed by allophanate hydrolase (13) to produce nance (13C-NMR). Sequence analysis revealed that this fungal 2 mol of ammonia and carbon dioxide, respectively. protein was most closely related to other predicted fungal genes in Current knowledge of cyanuric acid hydrolase genes and en- recently deposited genome sequences and that the fungal cyanuric zymes is derived exclusively from studies with bacterial strains (8, acid hydrolases are most closely related to bacterial cyanuric acid 10, 11, 14–16). A recent study found 41 homologs of known cya- hydrolases from the phylum Actinobacteria. nuric acid hydrolase genes in publicly available sequenced ge- nomes (11). In that study, only one eukaryotic cyanuric acid hy- drolase homolog, from the green alga Micromonas sp. strain Received 16 August 2013 Accepted 10 September 2013 RCC299, was found in the public databases, and it was not verified Published ahead of print 13 September 2013 to be a bona fide cyanuric acid hydrolase. Address correspondence to Lawrence P. Wackett, [email protected]. Other enzymes that are known to cleave cyclic amide bonds Supplemental material for this article may be found at http://dx.doi.org/10.1128 belong to large protein superfamilies (17), but the cyanuric acid /JB.00965-13. hydrolases are not evolutionarily related to any of these super- Copyright © 2013, American Society for Microbiology. All Rights Reserved. families and are relatively rare (11). Barbiturase catalyzes an anal- doi:10.1128/JB.00965-13 ogous ring-opening reaction with the pyrimidine barbituric acid December 2013 Volume 195 Number 23 Journal of Bacteriology p. 5233–5241 jb.asm.org 5233 Dodge et al. TABLE 1 Novel primers used in this study for PCR, RT-PCR, and tometer. Isolates were further characterized by rRNA gene sequencing. A sequencing 1,724-bp fragment from the nuclear small-subunit (18S) rRNA gene was Primer Sequence Characteristics amplified with primers NS1 and NS8 and sequenced with primers NS1, Glarea130F 5=-TGYGTYAAYGAYTTYTCTCGC-3= Degenerate PCR, forward NS2, NS7, and NS8 (27); a 541-bp fragment spanning internal transcribed Glarea509R 5=-GCCATAGAYTCRTADGTRTC-3= Degenerate PCR, reverse spacer 1 (ITS1), ITS2, and the 5.8S rRNA gene was amplified and se- SaroGSP1-5= 5=-TCAGGAGCGGGCATTTGATGA Genome walking, PCR, quenced with primers ITS1 and ITS4 (27). GAACCAT-3= forward DNA and protein sequence alignments and creation of phylogenetic SaroGSP1-3= 5=-TGCTGTAACAATATTCTCCGG Genome walking, PCR, trees. DNA sequences were aligned against the nucleotide collection in CGGCACT-3= reverse GenBank (Bethesda, MD) by using BLAST (28). Routine alignments of SaroGSP2-5= 5=-TGTTGAGCCTGTTCGTCTGT Genome walking, PCR, GCCAATCT-3= and sequencing, protein sequences were done with ClustalW (29). MEGA 5 (30) was used forward to align protein sequences with MUSCLE (31) and to generate maximum SaroGSP2-3= 5=-AGATTGGCACAGACGAACAG Genome walking, PCR, likelihood and neighbor-joining phylogenetic trees. A consensus maxi- GCTCAACA-3= and sequencing, mum likelihood tree was generated from 500 bootstrap replicates. The GI reverse Saro-1F 5=-ATGGCGCCGATTGAGATACTC-3= RT-PCR, forward numbers of the sequences used to create the phylogenetic trees are pre- Saro1-1026R 5=-CTATATACTGGTCTCGTACA RT-PCR, reverse sented in Table S1 in the supplemental material. CCAAAC-3= Anion-exchange chromatography to fractionate cyanuric acid Saro1-1047R 5=-TCATGATTTGTCTTCGGTGGAA-3= RT-PCR, reverse hydrolase activity. Sarocladium sp. strain CA was grown in 2,000 ml of NFB medium with 0.2% glucose and 3 mM cyanuric acid as the sole nitrogen source, harvested with a 150-ml, 0.2-␮m-mesh surfactant-free MATERIALS AND METHODS cellulose acetate (SFCA) membrane bottle-top filter (Nalgene, Rochester, NY), and ground for 5 min in a chilled mortar and pestle with 2 ml cold 20 Chemicals, media, and growth conditions. Commercial chemicals were mM Bis-Tris buffer (pH 7.0) (binding buffer) 1.0 g of 3M Empore Filter obtained from Sigma-Aldrich (St. Louis, MO), except for cyanuric acid Aid 400 high-density glass beads (Fisher Scientific, Pittsburgh, PA) per g (Alfa Aesar, Heysham, United Kingdom). Ammeline (2,4-diamino-6- wet biomass. The slurry was centrifuged in chilled microcentrifuge tubes hydroxy-1,3,5-triazine); ammelide (2-amino-4,6-dihydroxy-1,3,5-tria- at 14,000 rpm for 15 min at 4°C. Supernatants were passed through an zine); methyl-, 1,3-dimethyl-, and trimethylisocyanuric acids; 5-nitro Acrodisc 0.45-␮m HT Tuffryn membrane syringe filter (Pall Corpora- barbituric acid; and 5-methyl barbituric acid were synthesized in our lab- tion, Ann Arbor, MI), and the filtrate (5.5 ml) was mixed with 6.0 ml of a oratory as described previously (24, 25). Enrichment cultures and isolated DEAE-Sephadex A50 anion-exchange resin slurry in binding buffer. The strains were grown at 28°C in nitrogen-free basal salts (NFB) medium mixture was swirled periodically and kept on ice in a cold room for 1 h.
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