Discovery of a New Prokaryotic Type I GTP Cyclohydrolase Family

GTP cyclohydrolase I (GCYH-I) is the first enzyme of the de novo tetrahydrofolate biosynthetic pathway present in bacteria, fungi, and plants, and encoded in Escherichia coli by the folE gene. It is also the first enzyme of the biopterin (BH4) pathway in Homo sapiens, where it is encoded by a homologous folE gene. A homology-based search of GCYH-I orthologs in all sequenced bacteria revealed a group of microbes, including several clinically important pathogens, that encoded all of the enzymes of the tetrahydrofolate biosynthesis pathway but GCYH-I, suggesting that an alternate family was present in these organisms. A prediction based on phylogenetic occurrence and physical clustering identified the COG1469 family as a potential candidate for this missing enzyme family. The GCYH-I activity of COG1469 family proteins from a variety of sources (Thermotoga maritima, Bacillus subtilis, Acinetobacter baylyi, and Neisseria gonorrhoeae) was experimentally verified in vivo and/or in vitro. Although there is no detectable sequence homology with the canonical GCYH-I, protein fold recognition based on sequence profiles, secondary structure, and solvation potential information suggests that, like GCYH-I proteins, COG1469 proteins are members of the tunnel-fold (T-fold) structural superfamily. This new GCYH-I family is found in ∼20% of sequenced bacteria and is prevalent in Archaea, but the family is to this date absent in Eukarya. GTP cyclohydrolase I (GCYH-I) is the first enzyme of the de novo tetrahydrofolate biosynthetic pathway present in bacteria, fungi, and plants, and encoded in Escherichia coli by the folE gene. It is also the first enzyme of the biopterin (BH4) pathway in Homo sapiens, where it is encoded by a homologous folE gene. A homology-based search of GCYH-I orthologs in all sequenced bacteria revealed a group of microbes, including several clinically important pathogens, that encoded all of the enzymes of the tetrahydrofolate biosynthesis pathway but GCYH-I, suggesting that an alternate family was present in these organisms. A prediction based on phylogenetic occurrence and physical clustering identified the COG1469 family as a potential candidate for this missing enzyme family. The GCYH-I activity of COG1469 family proteins from a variety of sources (Thermotoga maritima, Bacillus subtilis, Acinetobacter baylyi, and Neisseria gonorrhoeae) was experimentally verified in vivo and/or in vitro. Although there is no detectable sequence homology with the canonical GCYH-I, protein fold recognition based on sequence profiles, secondary structure, and solvation potential information suggests that, like GCYH-I proteins, COG1469 proteins are members of the tunnel-fold (T-fold) structural superfamily. This new GCYH-I family is found in ∼20% of sequenced bacteria and is prevalent in Archaea, but the family is to this date absent in Eukarya. Folic acid, in the form of various tetrahydrofolate (THF) 4The abbreviations used are: THF, tetrahydrofolate; GCYH-I, GTP cyclohydrolase I; H2NTP, 7,8-dihydroneopterin triphosphate; dT, thymidine; Kan, kanamycin; HPLC, high pressure liquid chromatography; DHNA, 7,8-dihydroneopterin aldolase. derivatives, serves as a cofactor in one-carbon transfer reactions during the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (1Nichol C.A. Smith G.K. Duch D.S. Annu. Rev. Biochem. 1985; 54: 729-764Crossref PubMed Google Scholar). In bacteria THF is also involved in the biosynthesis of the initiator formylmethionyl-tRNA (2Clark B.F. Marcker K.A. J. Mol. Biol. 1966; 17: 394-406Crossref PubMed Scopus (145) Google Scholar). Plants, fungi, and most bacteria synthesize THF de novo from GTP and p-aminobenzoic acid (3Hanson A.D. Gregory J.F. II I Curr. Opin. Plant Biol. 2002; 5: 244-249Crossref PubMed Scopus (118) Google Scholar, 5Cossins E.A. Chen L. Phytochemistry. 1997; 45: 437-452Crossref PubMed Scopus (119) Google Scholar). Animals lack key enzymes of the folate biosynthetic pathway, and thus a dietary source of folate is required for normal growth and development (6Lucock M. Mol. Genet. Metab. 2000; 71: 121-138Crossref PubMed Scopus (657) Google Scholar). The folate pathway has a storied history as an important target in antibacterial therapeutics and cancer chemotherapy; dihydropteroate synthase is the target of the sulfonamides, the first synthetic drugs developed with broad-spectrum antibacterial activity (7Huovinen P. Sundström L. Swedberg G. Sköld O. Antimicrob. Agents Chemother. 1995; 39: 279-289Crossref PubMed Google Scholar), and dihydrofolate reductase is the target of methotrexate, the first anticancer chemotherapy developed. GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo THF pathway (1Nichol C.A. Smith G.K. Duch D.S. Annu. Rev. Biochem. 1985; 54: 729-764Crossref PubMed Google Scholar). It is encoded in Escherichia coli by the folE gene (8Katzenmeier G. Schmid C. Kellermann J. Lottspeich F. Bacher A. Biol. Chem. Hoppe-Seyler. 1991; 372: 991-997Crossref PubMed Scopus (34) Google Scholar, 9Schoedon G. Redweik U. Frank G. Cotton R.G. Blau N. Eur. J. Biochem. 1992; 210: 561-568Crossref PubMed Scopus (22) Google Scholar) and catalyzes a complex reaction (10Yim J.J. Brown G.M. J. Biol. Chem. 1976; 251: 5087-5094Abstract Full Text PDF PubMed Google Scholar) that begins with hydrolytic ring opening of the purine ring at C-8 to generate an N-formyl intermediate, which is then the site for a second hydrolysis with concomitant loss of C-8 as formic acid. In the subsequent steps of the reaction, the ribosyl moiety undergoes ring opening and an Amadori rearrangement followed by cyclization to generate the pterin ring in THF (Fig. 1). A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH4) pathway (Fig. 1), an essential cofactor for aromatic amino acid oxidation in the biosynthesis of tyrosine and neuro-transmitters, such as serotonin and 3,4-dihydroxy-l-phenylalanine (11Thony B. Auerbach G. Blau N. Biochem. J. 2000; 347: 1-16Crossref PubMed Scopus (729) Google Scholar, 12Bonafe L. Thony B. Penzien J.M. Czarnecki B. Blau N. Am. J. Hum. Genet. 2001; 69: 269-277Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Although several enzymes in the folate pathway have proven to be important antimicrobial targets (7Huovinen P. Sundström L. Swedberg G. Sköld O. Antimicrob. Agents Chemother. 1995; 39: 279-289Crossref PubMed Google Scholar), the presence of homologous GCYH-I enzymes in both humans and bacteria has precluded the development of GCYH-I as a viable target. The product of GCYH-I, 7,8-dihydroneopterin triphosphate (H2NTP), is subsequently dephosphorylated to 7,8-dihydroneopterin by both specific and nonspecific phosphatases (13Klaus S.M. Wegkamp A. Sybesma W. Hugenholtz J. Gregory 3rd, J.F. Hanson A.D. J. Biol. Chem. 2005; 280: 5274-5280Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and the remainder of THF biosynthesis is carried out by the enzymes encoded by the folBKPCA genes (in E. coli) (4Green J.C. Nichols B.P. Matthews R.G. Neidhart F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 665-673Google Scholar). Analysis of the distribution of the folate biosynthetic genes among sequenced organisms using the newly developed SEED data base (14Overbeek R. Begley T. Butler R.M. Choudhuri J.V. Chuang H.Y. Cohoon M. de Crécy-Lagard V. Diaz N. Disz T. Edwards R. Fonstein M. Frank E.D. Gerdes S. Glass E.M. Goesmann A. Hanson A. Iwata-Reuyl D. Jensen R. Jamshidi N. Krause L. Kubal M. Larsen N. Linke B. McHardy A.C. Meyer F. Neuweger H. Olsen G. Olson R. Osterman A. Portnoy V. Pusch G.D. Rodionov D.A. Ruckert C. Steiner J. Stevens R. Thiele I. Vassieva O. Ye Y. Zagnitko O. Vonstein V. Nucleic Acids Res. 2005; 33: 5691-5702Crossref PubMed Scopus (1507) Google Scholar) revealed that a large group of bacteria (Table 1) do not contain orthologs of folE while orthologs of all the other folate biosynthesis genes are present. We predicted that another protein family was responsible for the formation of H2NTP in these organisms, and we report here the combination of comparative genomic analysis and experimental validation that led to the identification of a new prokaryote-specific GCYH-I family.TABLE 1Distribution of FolE, COG1469, FolK, and FolP homologs in a subset of sequenced genomes The genomes used in the phylogenetic pattern search are in boldface type.OrganismFolECOG1469FolKFolPE. coli K12+++B. subtilis subsp. subtilis strain 168++++A. baylyi++++Bordetella bronchiseptica RB50+++Neisseria meningitidis MC58+++Neisseria europaea ATCC 19718+++Oceanobacillus iheyensis HTE831+++S. aureus subsp. aureus MW2+++T. maritima MSB8+++Desulfotalea psychrophila LSv54+++Desulfuromonas acetoxidans+++Exiguobacterium sp. 255-15+++Geobacter sulfurreducens PCA+++Magnetococcus sp. MC-1+++Methylobacillus flagellatus KT+++Neisseria lactamica ST-640+++Neisseria meningitidis FAM18+++Silicibacter sp. TM1040 (B)+++Rhodobacter sphaeroides 2.4.1+++N. gonorrhoeae FA 1090+++Geobacter metallireducens GS-15+++Idiomarina loihiensis L2TR+++Oceanicola batsensis HTCC2597+++Rhodobacterales bacterium HTCC2654+++Staphylococcus haemolyticus JCSC1435+++Thiomicrospira crunogena XCL-2+++Sulfitobacter sp. EE-36+++Roseovarius nubinhibens ISM (B)+++Staphylococcus saprophyticus+++Loktanella vestfoldensis SKA53+++Roseobacter sp. MED193+++Desulfovibrio vulgaris+++ Open table in a new tab Bioinformatics—Analysis of the folate subsystem was performed in the SEED data base (14Overbeek R. Begley T. Butler R.M. Choudhuri J.V. Chuang H.Y. Cohoon M. de Crécy-Lagard V. Diaz N. Disz T. Edwards R. Fonstein M. Frank E.D. Gerdes S. Glass E.M. Goesmann A. Hanson A. Iwata-Reuyl D. Jensen R. Jamshidi N. Krause L. Kubal M. Larsen N. Linke B. McHardy A.C. Meyer F. Neuweger H. Olsen G. Olson R. Osterman A. Portnoy V. Pusch G.D. Rodionov D.A. Ruckert C. Steiner J. Stevens R. Thiele I. Vassieva O. Ye Y. Zagnitko O. Vonstein V. Nucleic Acids Res. 2005; 33: 5691-5702Crossref PubMed Scopus (1507) Google Scholar) with SEED version cvs.1144925141 (05:45:41 on April 13, 2006) (available on the World Wide Web at anno-3.nmpdr.org/anno/FIG/index.cgi). Results are made available in the “Folate Biosynthesis Subsystem” on the publicly available server (available on the World Wide Web at theseed.uchicago.edu/FIG/index.cgi). The phylogenetic pattern search was performed on the following SEED server: www.nmpdr.org/FIG/sigs.cgi?SPROUT=1. Strains and Growth Conditions—Bacteria were routinely grown in LB medium (BD Diagnostic Systems) at 37 °C. Growth media were solidified with 15 g/liter agar (BD Diagnostic Systems) for the preparation of plates. Transformations and P1 transductions were performed following standard procedures (15Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar, 16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Thymidine (dT; 300 μm), ampicillin (100 μg/ml), kanamycin (Kan; 50 μg/ml), and were as was with a was carried out with liquid was on a liquid and was on a an with an source at the in the of and at and of maritima, Neisseria and Bacillus subtilis COG1469 for COG1469 genes from T. maritima N. gonorrhoeae and B. subtilis were by from genomic of the organisms. The following to the and of the were used for T. maritima T. maritima N. gonorrhoeae N. gonorrhoeae B. subtilis and B. subtilis in boldface are the and and in the is the The of genomic 50 of the and by the and of in a of 50 A was 1) for of at for at 50 for and at for for The product was from a using the and a The of the and were by of the T. maritima, Acinetobacter baylyi, and B. subtilis COG1469 for the gene A. E. J. 2005; PubMed Scopus Google Scholar) was a of the for The COG1469 genes from B. subtilis and A. were in D. J. J. 1995; PubMed Scopus Google Scholar). The following to the and of the gene were B. subtilis B. subtilis A. and A. were and as and then with D. J. J. 1995; PubMed Scopus Google Scholar) with the and The of the A. and B. subtilis were by as as and were the E. coli a of Hanson of S.M. de A. S. F. Gregory J.F. Hanson A.D. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). The were on LB with dT, and and for the to on LB in the presence of various of the presence of the and of the in the the were by using the following and from the folE gene and and of the in the and and of T. maritima, N. and B. subtilis COG1469 and were E. coli for of of the were grown at 37 with an of was was to a of and the were for an at with The were by at for at °C. The was in liquid and at was and in 50 and at a of The were by the of and to a of and The was at for at and the was protein The was an that with A (100 300 and The was with of of (100 300 and and of (100 300 and The protein was from the with of The protein was in a and at 50 50 and The was from the T. maritima, N. and B. subtilis COG1469 proteins in reactions that protein 50 in a of for at the reactions were a of in A. protein was from the with of A. The protein was and 50 50 and of acid from was to the of J. J.J. J. PubMed Scopus Google Scholar) and Brown G.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were performed at 37 N. B. in and enzyme in a of of 50 were at specific and by the of of formic acid. on for the were a of that in 50 was from the with 50 and the was and by liquid were performed in and E. coli FolE, COG1469 enzyme from B. subtilis N. gonorrhoeae in a of The reactions were to for at 37 while the from to A. N. Bacher A. 2001; PubMed Scopus Google Scholar). were performed in and E. coli COG1469 enzyme in a of were with the of GTP to a of and for at 37 N. B. was and the reaction were for at 37 °C. the reactions was then of a in and the were at in the for 15 was by the of of acid. The were then by at and at were performed in and E. coli COG1469 protein in were at 37 for followed by the of and the reactions were an at 37 °C. The reaction were by on a in The was developed at with the following Analysis of GCYH-I preparation of from GCYH-I reaction for liquid analysis was carried out in reaction and E. coli COG1469 from N. gonorrhoeae in a of The reaction were at 37 for in the and with in a of the of and Brown (10Yim J.J. Brown G.M. J. Biol. Chem. 1976; 251: 5087-5094Abstract Full Text PDF PubMed Google Scholar). the reactions at for in the the were a The was with of of of and of The from the was with acid. The were in liquid and to and then in and and The were by with an with an The was in the using the following of and of and of 50 of and of °C. was to the of and by at a of The was in Analysis of genes of the de novo folate pathway are and Hanson and V. de which dihydropteroate synthase and organisms that these genes have a of the folE of the from 7,8-dihydroneopterin triphosphate to are in bacteria O. 2000; PubMed Scopus Google Scholar). Analysis of the distribution of the folE gene among all sequenced genomes that homologs revealed a large of organisms (Table and 1) that folE suggesting that folE was P. Genet. PubMed Scopus Google Scholar) in these organisms. a SEED that identification of protein that a phylogenetic distribution we the available genomes for protein that were present in organisms that lack folE homologs (Table in boldface and absent in E. protein phylogenetic of COG1469, was of and as in members of this family with folate genes in several organisms. 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PubMed Scopus Google Scholar). the E. coli strain has a growth on of to form at 37 to the of of the initiator The strain was with the COG1469 from T. maritima Although of both the not and the growth was (Fig. it was not and on high This is not T. maritima is a and of the enzymes from activity at 37 °C. the COG1469 orthologs from the B. subtilis and A. as Acinetobacter sp. were and the E. coli of (Fig. and growth (Fig. was with these and is with COG1469 family proteins GCYH-I COG1469 GCYH-I in with the in vivo COG1469 genes were protein to of the of GTP cyclohydrolase I activity with in of the genes COG1469 proteins from T. maritima, N. and B. subtilis were from genomic the and the proteins were and of the proteins were as enzymes both as the and the using (10Yim J.J. Brown G.M. J. Biol. 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