Biosynthesis of Pteridines in Escherichia coli

Abstract

An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate. The gene was subsequently designated folB. The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on theE. coli chromosome. The folX andfolB gene products were both expressed to high yield in recombinant E. coli strains, and the recombinant proteins were purified to homogeneity. Both enzymes form homo-octamers. Aldolase can use l-threo-dihydroneopterin andd-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2′ of dihydroneopterin and dihydromonapterin at appreciable velocity. Epimerase catalyzes the epimerization of carbon 2′ in the triphosphates of dihydroneopterin and dihydromonapterin. However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate. Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions. We propose that the polarization of the 2′-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity. A deletion mutant obtained by targeting the folX gene of E. coli has normal growth properties on complete medium as well as on minimal medium. Thus, the physiological role of the E. coli epimerase remains unknown. The open reading frameygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase. However, the genome of H. influenzae does not specify a dihydroneopterin-triphosphate epimerase. An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate. The gene was subsequently designated folB. The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on theE. coli chromosome. The folX andfolB gene products were both expressed to high yield in recombinant E. coli strains, and the recombinant proteins were purified to homogeneity. Both enzymes form homo-octamers. Aldolase can use l-threo-dihydroneopterin andd-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2′ of dihydroneopterin and dihydromonapterin at appreciable velocity. Epimerase catalyzes the epimerization of carbon 2′ in the triphosphates of dihydroneopterin and dihydromonapterin. However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate. Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions. We propose that the polarization of the 2′-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity. A deletion mutant obtained by targeting the folX gene of E. coli has normal growth properties on complete medium as well as on minimal medium. Thus, the physiological role of the E. coli epimerase remains unknown. The open reading frameygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase. However, the genome of H. influenzae does not specify a dihydroneopterin-triphosphate epimerase. The pathway of tetrahydrofolate biosynthesis is an important drug target in bacteria and protozoal parasites (1Achari A. Somers D.O. Champness J.N. Bryant P.K. Rosemond J. Stammers D.K. Nat. Struct. Biol. 1997; 4: 490-497Crossref PubMed Scopus (237) Google Scholar, 2Fleischmann R.D. et al.Science. 1995; 269: 496-512Crossref PubMed Scopus (4694) Google Scholar, 3Spitzy K.H. Infection. 1991; 19: 52-56Crossref PubMed Scopus (2) Google Scholar). Thus, the sulfonamides inhibiting dihydropteroate synthase were the first synthetic agents that could be used with high efficiency against a wide variety of microbial and protozoal parasites. Even after more than 60 years, they remain important therapeutic agents (1Achari A. Somers D.O. Champness J.N. Bryant P.K. Rosemond J. Stammers D.K. Nat. Struct. Biol. 1997; 4: 490-497Crossref PubMed Scopus (237) Google Scholar, 4Chio L.C. Bolyard L.A. Nasr M. Queener S.F. Antimicrob. Agents Chemother. 1996; 40: 727-733Crossref PubMed Google Scholar). More recently, trimethoprim was found to act as an inhibitor of bacterial dihydrofolate reductase (5Garg S.K. Ghosh S.S. Mathur V.S. Int. J. Clin. Pharmacol. Ther. Toxicol. 1986; 24: 23-25PubMed Google Scholar). The combined application of trimethoprim and a sulfonamide is an important protocol for the treatment of bacterial infections (5Garg S.K. Ghosh S.S. Mathur V.S. Int. J. Clin. Pharmacol. Ther. Toxicol. 1986; 24: 23-25PubMed Google Scholar, 6Fihn S.D. Johnson C. Roberts P.L. Running K. Stamm W.E. Ann. Intern. Med. 1988; 108: 350-357Crossref PubMed Scopus (76) Google Scholar, 7Hughes W.T. Killmar J. Antimicrob. Agents Chemother. 1996; 40: 962-965Crossref PubMed Google Scholar, 8Norrby S.R. Rev. Infect. Dis. 1990; 12: 458-467Crossref PubMed Scopus (178) Google Scholar). Tetrahydrofolate is biosynthesized by plants and many microorganisms. The first committed step catalyzed by GTP cyclohydrolase I is a mechanistically complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate (Compound 1) (Fig. 1) (9Nar H. Huber R. Auerbach G. Fischer M. Hösl C. Ritz H. Bracher A. Meining W. Eberhardt S. Bacher A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12120-12125Crossref PubMed Scopus (103) Google Scholar). A pyrophosphatase and a phosphatase have been proposed to convert Compound 1 to dihydroneopterin (Compound 3) in two consecutive steps (10Suzuki Y. Brown G.M. J. Biol. Chem. 1974; 249: 2405-2410Abstract Full Text PDF PubMed Google Scholar), but firm evidence for the involvement of specific enzymes in the conversion of Compound 1 to Compound 3 has not been obtained. Recently, it has been proposed that the release of pyrophosphate from Compound 1 can be catalyzed of two-valent ions such as Mg2+ and Ca2+ (11De Saizieu A. Vankan P. van Loon A.P.G.M. Biochem. J. 1995; 306: 371-377Crossref PubMed Scopus (36) Google Scholar). The conversion of Compound 3 to 6-hydroxymethyl-7,8-dihydropterin (Compound 4) is catalyzed by dihydroneopterin aldolase (12Mathis J.B. Brown G.M. Methods Enzymol. 1980; 66: 556-560Crossref PubMed Scopus (8) Google Scholar). Compound 4 is converted to dihydrofolate by the subsequent condensation with 4-aminobenzoate and glutamate by the catalytic action of dihydropteroate synthase and dihydrofolate synthetase, respectively (13Green J.M. Nichols B.P. Matthews R.G. Neidhardt F. Escherichia coli and Salmonella typhimurium. 2nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 665-673Google Scholar). An enzyme catalyzing the epimerization of the folate precursor, dihydroneopterin triphosphate, under formation of 7,8-dihydromonapterin (Compound 2) had been observed in Escherichia coli by Heine and Brown (14Heine M.C. Brown G.M. Biochim. Biophys. Acta. 1975; 411: 236-240Crossref PubMed Scopus (13) Google Scholar). Recently, the primary structure of this enzyme has been determined and has been found to show similarity to the dihydroneopterin aldolase domain of a multifunctional folate biosynthetic enzyme from Pneumocystis carinii (15Haußmann C. Rohdich F. Lottspeich F. Eberhardt S. Scheuring J. Mackamul S. Bacher A. J. Bacteriol. 1997; 179: 949-951Crossref PubMed Google Scholar). On the other hand, the epimerase is similar to a reading frame at 69.0 kb 1The abbreviations used are: kb, kilobase(s); PIPES, 1,4-piperazinediethanesulfonic acid; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; bp, base pair(s). of the E. colichromosome designated ygiG (EMBL accession number L12966) in the close vicinity of the bacitracin resistance gene. This reading frame specifies the dihydroneopterin aldolase of E. coli,which is a potential target for the development of enzyme inhibitors with antibacterial and/or antiprotozoal activity. This paper reports on structural and mechanistic similarities of dihydroneopterin-triphosphate epimerase and dihydroneopterin aldolase. It also shows that the single Hemophilus influenzae homolog of these proteins (specified by the unannotated open reading frameygiG_haein) is an aldolase-type enzyme. 6-Hydroxymethylpterin,d-dihydroneopterin, l-dihydromonapterin,d-dihydromonapterin, l-neopterin,l-monapterin, d-dihydroneopterin monophosphate,d-neopterin, and dihydrobiopterin were purchased from Dr. B. Schircks (Jona, Switzerland). Synthetic oligonucleotides were purchased from MWG (Ebersberg, Germany). Restriction enzymes and T4 DNA ligase were obtained from Pharmacia (Freiburg, Germany), Life Technologies, Inc. (Eggenstein, Germany), and New England Biolabs Inc. (Beverly, MA). Goldstar DNA polymerase was obtained from Eurogentec (Seraing, Belgium). GTP cyclohydrolase I was purified from a recombinant E. coli strain as described earlier (9Nar H. Huber R. Auerbach G. Fischer M. Hösl C. Ritz H. Bracher A. Meining W. Eberhardt S. Bacher A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12120-12125Crossref PubMed Scopus (103) Google Scholar). The bacterial strains and plasmids used in this study are summarized in TableI.Table IStrains and plasmids used in this studyStrain or plasmidGenotype or relevant characteristicRef. or sourceE. coli DSM613Wild typeDSM Marburg XL1-BluerecA1,endA1,gyrA96,thi-1,hsdR17,supE44,relA1,lac[F′,proAB,lacIqZΔM15,Tn10(tet′)]31Bullock W.O. Fernandez J.M. Short J.M. BioTechniques. 1987; 5: 376-379Google Scholar M15(pREP4)lac,ara,gal,mtl,recA+,uvr+[pREP4,lacI,kanar]32Zamenhof P.J. Villarejo M. J. Bacteriol. 1972; 110: 171-178Crossref PubMed Google Scholar SK6600F−,ara−,Δ(lac-proAB),rpsL,F80,lacZΔM15 (r+,m+)17Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar SK6600E0F−,ara−,Δ(lac-proAB),ΔfolX,rpsL,F80,lacZΔM15(r+,m+) (kanar)This studyPlasmids pBluescript SKII−Stratagene pNCO113High copy number plasmid vector27Stüber D. Matile H. Garotta G. Lefkovits F. Pernis P. Immunological Methods. IV. Academic Press, San Diego1990: 121-152Google Scholar GHIFL01pUC18 with a 4664-bp fragmentATCC Bepi213pBluescript SK11− with a 213-bp fragmentThis study pEPIpNCO113 with a 375-bp fragmentThis study pEALpNCO113 with a 400-bp fragmentThis study pHIALpNCO113 with a 392-bp fragmentThis study pMAK705Low copy number plasmid vector with temperature-sensitive origin for DNA replication (Cmr)17Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar pMAK705EpMAK705 with a 1000-bp fragmentThis study pMAK705EKpMAK705 with a 1800-bp fragment (kanar)This study Open table in a new tab The epimerase substrate, dihydroneopterin triphosphate, was produced enzymatically from GTP. Reaction mixtures containing 100 mm Tris-HCl, pH 7.5, 0.1m KCl, 50 mm GTP, 5 mmdithiothreitol, and 0.05 mg/ml recombinant E. coli GTP cyclohydrolase I were incubated for 4 h at 37 °C. Protein was then removed by ultrafiltration using a YM-30 membrane (Amicom Inc.). An aliquot of this reaction mixture (20 μl) was added to 80 μl of a solution containing 80 mm PIPES, pH 6.2, and 8 mm MgCl2. The epimerase reaction was started by the addition of protein solution. The reaction mixture was incubated at 55 °C for 10 min. The reaction was terminated by the addition of a solution (30 μl) containing 1% I2 and 2% (w/v) KI in 1m HCl. The samples were incubated at room temperature for 5 min. Excess iodine was reduced by the addition of 2% ascorbic acid (10 μl, w/v). A solution (210 μl) containing 1.5 mTris-HCl, pH 8.5, 4.8 mm ZnCl2, 4.5 mm EDTA, and 0.5 units of alkaline phosphatase (Boehringer Mannheim, grade II) was added. The mixture was incubated at 37 °C for 90 min. Trichloroacetic acid (50 μl, 40%, w/v) was added. Aliquots of 10 μl were analyzed by using a of and an containing (w/v) pH The was The was The was and The is on a described earlier (12Mathis J.B. Brown G.M. Methods Enzymol. 1980; 66: 556-560Crossref PubMed Scopus (8) Google Scholar). mixtures 100 mm Tris-HCl, pH 5 mm and The mixture was incubated at °C for 10 min. The reaction was terminated by the addition of μl of 1 containing 1% I2 and 2% (w/v) The samples were incubated at room temperature for 5 min. Excess iodine was reduced by the addition of 2% ascorbic acid (10 μl, w/v). The samples were analyzed by using a of and an containing mm and was The was The plasmids for the folX andfolB were as described by et G. Fischer M. C. Eberhardt S. H. Bacher A. J. Bacteriol. 1997; 179: PubMed Google Scholar). The folX and coli were by with DNA coli as The gene influenzae was by using the plasmid as The folX gene was with the oligonucleotides as and as The of E. coli was with as and as and the gene influenzae was with as and as as for a with the and The was used for the folX gene. The was used for the gene of E. and the for the gene of H. The products influenzae and folX of E. were with and and in the vector which had been and the gene of E. coli was with and then the vector which had been with and The plasmids designated the of E. the gene of E. and containing gene of H. were E. coli (20 and were added for the of the plasmids in the used for Open table in a new tab of plasmids for the of E. coli and H. influenzae and for the folX gene of E. 1 for for for for Open table in a new tab The folX gene and the were by with as and as II) using E. coli DNA as The was with and and then the vector which had been with and The vector the temperature-sensitive origin of replication and a resistance gene. The was with The folX gene two and in the of a DNA fragment of A resistance was by using the and the II) with the plasmid as The plasmid was and the was with the resistance the plasmid The of the folX gene is a of the described by et C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar), and M. P. J. Bacteriol. PubMed Google Scholar), and and C. G. J. Bacteriol. 1997; 179: PubMed Google Scholar). The plasmid was E. coli strain The plasmid at but not at °C. at °C on that with the plasmid the chromosome by This in a of the folX gene to the folX gene with the was using two of in to the of the had by The to the E. coli chromosome in the vicinity of the of the folX in with the to the in a fragment of The to the and the to the E. coli chromosome in the vicinity of the of the folX in the of a of with this the of The of this was by of the bacterial strain in liquid medium containing at °C. were then and on containing at °C. of the that of the an folX gene on the plasmid and the folX gene on the chromosome. of the was by at °C on The were to Epimerase could not be in of the using the and the II) in of bp, the folX gene of the of growth with or folX were on medium as well as on minimal medium with E. coli strain the plasmid was to a of units in with medium containing and at 37 °C. was added to a of and was for a of 6 were by with 50 mm Tris-HCl, pH and at °C. was in 50 of 50 pH containing mg/ml and mg/ml were by and was removed by The was to °C in a for and was then in The was removed by The was on a that had been with mm Tris-HCl, pH The was with a of were combined and by ultrafiltration were purified by on a 60 The was with mm Tris-HCl, pH containing 100 mm at a of 3 aldolase from H. influenzae could be purified using the E. the plasmid was in medium containing and The was incubated at 37 °C with a of was added to a of and was The were by and at °C. was and in 80 of 50 mm Tris-HCl, pH containing mg/ml and mg/ml were by The was and the was on a The was with a of containing mm Tris-HCl, pH were by ultrafiltration and to treatment at 80 °C for 4 min. The was removed by The enzyme was purified by on a 60 The was with pH containing 100 mm The was 3 Steady-state kinetic were at 37 °C. were determined by to the using the and The for epimerization of dihydroneopterin triphosphate and dihydromonapterin triphosphate catalyzed by dihydroneopterin-triphosphate epimerase was from the of reaction mixtures that had a of were with an with and were in with and (10 was added to the for The specific was on the of acid in and Society of Scholar). Protein in 100 mm pH was at 4 °C and for Protein was by We have reported the of dihydroneopterin-triphosphate epimerase from E. coli (15Haußmann C. Rohdich F. Lottspeich F. Eberhardt S. Scheuring J. Mackamul S. Bacher A. J. Bacteriol. 1997; 179: 949-951Crossref PubMed Google Scholar). A base that an open reading frame in the of the located at 69.0 kb on the E. coli chromosome specifies a similar protein acid This open reading which was subsequently designated also shows similarity to dihydroneopterin from a variety of (Fig. 3) R.D. et al.Science. 1995; 269: 496-512Crossref PubMed Scopus (4694) Google Scholar, S. H. A. E. Y. M. M. S. A. A. K. S. S. C. A. M. M. S. DNA 1996; PubMed Scopus Google Scholar, J. Bacteriol. 1990; PubMed Scopus Google Scholar, J. J. Bacteriol. 1990; PubMed Google Scholar, 1997; PubMed Scopus Google Scholar, S. K. B. S. Proc. Natl. Acad. Sci. U. S. A. 1990; Scopus Google Scholar, B. P. J. Bacteriol. 1995; PubMed Google F. 1995; PubMed Scopus Google Scholar). J. and J. H. accession number The gene of E. coli was by with DNA and under the of the and a in the plasmid A coli strain this plasmid expressed a as shown by of the recombinant strain had a dihydroneopterin aldolase of as with a of in the E. coli strain This for the that gene for dihydroneopterin aldolase. The recombinant protein was purified by as described under A is summarized in IV. The purified protein as a single on The of that the protein had been expressed to a of of protein in the recombinant E. of recombinant dihydroneopterin aldolase from E. formation of from (Compound 3) was at The formation of from (Compound 3) was at °C. Open table in a new tab The H. influenzae genome open reading frame located at kb on the that is similar to the gene of E. coli acid The H. influenzae gene was expressed in a recombinant E. coli strain and purified in the as the E. coli aldolase. on the of the the recombinant protein catalyzed the cleavage of The kinetic parameters were similar to of the E. coli aldolase. The folX gene of E. coli dihydroneopterin-triphosphate epimerase was expressed under the of the and The recombinant protein was purified by a of and The purified protein as a single of on of recombinant dihydroneopterin-triphosphate epimerase from E. formation of dihydromonapterin triphosphate from dihydroneopterin triphosphate was at 55 The formation of dihydromonapterin triphosphate from dihydroneopterin triphosphate was at 55 °C. Open table in a new tab The of the proteins under study were determined by The are summarized in and that the proteins under study form of the enzymes from E. on acid on acid by on acid by Open table in a new tab of the similarity epimerase and aldolase, it was in to a of catalytic and kinetic enzyme were at the temperature of the enzymes °C for dihydroneopterin aldolase and 55 °C for dihydroneopterin-triphosphate and The more kinetic reported in were at 37 of epimerase and aldolase at 37 of coli dihydroneopterin aldolase 7,8-dihydroneopterin 7,8-dihydromonapterin influenzae dihydroneopterin aldolase coli dihydroneopterin-triphosphate epimerase 7,8-dihydroneopterin 7,8-dihydromonapterin Open table in a new tab were determined by after and as The shown in and 4 that the specified by the of E. H. influenzae catalyze the cleavage of The was in with as determined by and Brown (12Mathis J.B. Brown G.M. Methods Enzymol. 1980; 66: 556-560Crossref PubMed Scopus (8) Google Scholar). The can be by the pH at which and Brown determined kinetic were at pH but have found a in at pH However, the enzyme could also catalyze the epimerization of position of dihydromonapterin. The of the and were found that the dihydromonapterin could also serve as a substrate for cleavage of the side cleavage and epimerization catalyzed by the enzyme have similar The kinetic properties of the aldolase influenzae are shown in in The substrate of the in the biosynthetic pathway of tetrahydrofolate is However, the dihydromonapterin can be converted to epimerization to dihydroneopterin as the of the cleavage reaction for both is at than the epimerization reaction of both substrates It be that the gene of E. coli catalyzes the epimerization reaction more then the H. influenzae that in H. the of as well as the of the formation of from is as with the E. coli enzyme. The of dihydroneopterin can be by the of E. coli and H. but the is as with the The triphosphates of dihydroneopterin and dihydromonapterin were by the under shown in and dihydroneopterin-triphosphate epimerase the triphosphates of dihydroneopterin and dihydromonapterin as substrates for A of was for the on the of the kinetic and dihydromonapterin can also serve as substrates for but the reaction of the substrates are by more than a However, the substrates are also converted to The of the aldolase-type reaction catalyzed by epimerase is as with the cleavage of dihydroneopterin by the FolB The cleavage be to a of recombinant dihydroneopterin aldolase with dihydroneopterin-triphosphate epimerase specified by the folX gene aldolase and epimerase are by the step at a of aldolase at The physiological role of the FolB protein in the pathway of tetrahydrofolate biosynthesis is well However, the role of the epimerase in E. but not in H. is not We deletion mutant by gene targeting in E. the described by Kushner and C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). The of the plasmids and the gene deletion are described under of DNA that the gene of the mutant an of after of the of the mutant are of epimerase activity. the enzyme used is the have been as with theE. coli The folX mutant normal growth properties on minimal medium as well as on complete medium. Thus, the role of epimerase remains unknown. The enzymes specified by the folX and of E. coli are similar in of primary structure and The similarity to dihydroneopterin of several other (Fig. of these proteins are proteins catalyzing several steps in the biosynthesis of tetrahydrofolate. It to that the gene is in the pathway of tetrahydrofolate it is for the of the position 6 side chain of the pteridine On the other hand, the role of the dihydroneopterin-triphosphate epimerase is unknown. of E. coli with a folX gene with the of the side chain that is by the epimerization reaction have been found in a variety of H. G. K. Biol. Chem. 1989; Scholar, G. J. Biol. Chem. Full Text PDF PubMed Google Scholar), but role is unknown. Even these have a specific a specific epimerase not be for formation at in E. coli and H. they can be by the aldolase specified by gene. H. influenzae has a gene unannotated reading frame a protein catalytic properties are similar to of the FolB protein of E. However, the of H. influenzae and to be a pteridine epimerase, in with the that the epimerase is not an protein in A reaction is shown in We propose that both reaction can be by of by at the of or as well as aldolase-type can be catalyzed by both the FolB and This the of a for both of A cleavage of the and is proposed to be the reaction step for both enzyme reactions. at from of the cleavage reaction The of and proteins could from in the from the could the reaction products from cleavage of the of from the under of a the reaction for cleavage and could then on the of the cleavage at the This that in the of epimerase, the release of the cleavage products from the enzyme could be by the protein and release after of the cleavage We for with the of this Dr. A. P. G. van Loon for bacterial strains and and Dr. for