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• W2019965297 abstract "The early steps in the biosynthesis of the molybdopterin portion of the molybdenum cofactor have been investigated through the use of radiolabeled precursors. Labeled guanosine was added to growing cultures of the molybdopterin-deficient Escherichia coli mutant, moeB, which accumulates large amounts of precursor Z, the final intermediate in molybdopterin biosynthesis (Wuebbens, M. M., and Rajagopalan, K. V. (1993) J. Biol. Chem. 268, 13493-13498). Precursor Z is readily oxidized to the stable, fluorescent pterin, compound Z, which contains all 10 of the carbon atoms present in molybdopterin. For these experiments, compound Z was isolated from both the cells and culture media and analyzed for the presence of label. The development of a method for sequential cleavage of the compound Z side chain carbons facilitated determination of the distribution of label between the ring and the side chain of compound Z. Addition of uniformly labeled [14C]guanosine to moeB cultures produced compound Z labeled in both the ring and the side chain. Growth on [8-14C]guanosine resulted in transfer of label to the C-1′ position of compound Z. The label present in compound Z purified from cultures grown on [8,5′-3H]guanosine was lost by removal of the three terminal side chain carbons. These results indicate that although a guanosine compound serves as the initial precursor for molybdopterin biosynthesis, the early steps of this pathway in E. coli proceed via a pathway unlike that of any known pteridine biosynthetic pathway. The early steps in the biosynthesis of the molybdopterin portion of the molybdenum cofactor have been investigated through the use of radiolabeled precursors. Labeled guanosine was added to growing cultures of the molybdopterin-deficient Escherichia coli mutant, moeB, which accumulates large amounts of precursor Z, the final intermediate in molybdopterin biosynthesis (Wuebbens, M. M., and Rajagopalan, K. V. (1993) J. Biol. Chem. 268, 13493-13498). Precursor Z is readily oxidized to the stable, fluorescent pterin, compound Z, which contains all 10 of the carbon atoms present in molybdopterin. For these experiments, compound Z was isolated from both the cells and culture media and analyzed for the presence of label. The development of a method for sequential cleavage of the compound Z side chain carbons facilitated determination of the distribution of label between the ring and the side chain of compound Z. Addition of uniformly labeled [14C]guanosine to moeB cultures produced compound Z labeled in both the ring and the side chain. Growth on [8-14C]guanosine resulted in transfer of label to the C-1′ position of compound Z. The label present in compound Z purified from cultures grown on [8,5′-3H]guanosine was lost by removal of the three terminal side chain carbons. These results indicate that although a guanosine compound serves as the initial precursor for molybdopterin biosynthesis, the early steps of this pathway in E. coli proceed via a pathway unlike that of any known pteridine biosynthetic pathway. INTRODUCTIONWith the exception of nitrogenase, the molybdenum atom in all molybdoenzymes from animals, plants, and microorganisms is part of an organometallic structure termed the molybdenum cofactor. The extreme lability of the free cofactor following release from the molybdoenzymes has precluded both its complete purification and its direct chemical characterization. However, from structural analysis of three inactive derivatives of the cofactor (form A, form B, and dicarboxamidomethylmolybdopterin), the dithiolene-containing pterin structure shown in Fig. 1, termed molybdopterin, has been proposed to be the organic moiety of the cofactor from sulfite oxidase(1Johnson J.L. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6856-6860Crossref PubMed Scopus (268) Google Scholar, 2Johnson J.L. Hainline B.E. Rajagopalan K.V. Arison B.H. J. Biol. Chem. 1984; 259: 5414-5422Abstract Full Text PDF PubMed Google Scholar, 3Kramer S.P. Johnson J.L. Ribeiro A.A. Millington D.S. Rajagopalan K.V. J. Biol. Chem. 1987; 262: 16357-16363Abstract Full Text PDF PubMed Google Scholar). It is now known that molybdopterin is also the essential component of a family of dinucleotide variants of the cofactor which contain a nucleoside monophosphate linked to the terminal phosphate of the pterin (4Rajagopalan K.V. Johnson J.L. J. Biol. Chem. 1992; 267: 10199-10202Abstract Full Text PDF PubMed Google Scholar). In addition, the isomeric state of the dihydro pterin ring of the active cofactor may vary from enzyme to enzyme(5Gardlik S. Rajagopalan K.V. J. Biol. Chem. 1990; 265: 13047-13054Abstract Full Text PDF PubMed Google Scholar, 6Gardlik S. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 4889-4895Abstract Full Text PDF PubMed Google Scholar, 7Gardlik S. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 16627-16632Abstract Full Text PDF PubMed Google Scholar).Exploration of the pathway of biosynthesis of the molybdenum cofactor has been facilitated by the existence of pleiotropic mo mutants (8Shanmugam K.T. Stewart V. Gunsalus R.P. Boxer D.H. Cole J.A. Chippaux M. DeMoss J.A. Giordano G. Lin E.C.C. Rajagopalan K.V. Mol. Microbiol. 1992; 6: 3452-3454Crossref PubMed Scopus (68) Google Scholar) in a variety of organisms. Since these mutations result in loss of the activities of all molybdoenzymes in an organism, the proteins encoded at the mo loci are presumably involved in the synthesis of functional cofactor. In Escherichia coli, such mutants are chlorate-resistant, and recent studies involving a number of these chl mutants (now termed mo; (8Shanmugam K.T. Stewart V. Gunsalus R.P. Boxer D.H. Cole J.A. Chippaux M. DeMoss J.A. Giordano G. Lin E.C.C. Rajagopalan K.V. Mol. Microbiol. 1992; 6: 3452-3454Crossref PubMed Scopus (68) Google Scholar) have clarified the final steps of cofactor biosynthesis in this organism. In the terminal step of molybdopterin synthesis, the desulfo molybdopterin intermediate precursor Z (9Johnson J.L. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1989; 264: 13440-13447Abstract Full Text PDF PubMed Google Scholar, 10Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13493-13498Abstract Full Text PDF PubMed Google Scholar) is converted to molybdopterin through the action of molybdopterin synthase (previously termed “converting factor”)(11Pitterle D.M. Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13506-13509Abstract Full Text PDF PubMed Google Scholar, 12Pitterle D.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13499-13505Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 1, this conversion involves the opening of the cyclic phosphate ring of precursor Z, as well as the addition of two side chain sulfhydryl groups. No other small molecules are required for the reaction, and it appears that the sulfurs are covalently attached to molybdopterin synthase itself prior to their transfer to precursor Z(11Pitterle D.M. Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13506-13509Abstract Full Text PDF PubMed Google Scholar, 12Pitterle D.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13499-13505Abstract Full Text PDF PubMed Google Scholar). Addition of GMP to form the molybdopterin guanine dinucleotide form of the pterin present in E. coli is then mediated by the mob gene product(s)(13Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar).The dihydropterin, precursor Z, is labile and is readily converted to the stable pterin, compound Z, by air or iodine oxidation. As shown in Fig. 1, compound Z differs from precursor Z only in the reduction state of its pterin ring(10Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13493-13498Abstract Full Text PDF PubMed Google Scholar). Precursor Z accumulates in the E. coli mutants moeB and moaE(9Johnson J.L. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1989; 264: 13440-13447Abstract Full Text PDF PubMed Google Scholar, 10Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13493-13498Abstract Full Text PDF PubMed Google Scholar, 14Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 110-116Crossref PubMed Google Scholar) and is also present in the urine of group B cofactor-deficient humans(15Johnson J.L. Wuebbens M.M. Mandell R. Shih V.E. J. Clin. Invest. 1989; 83: 897-903Crossref PubMed Scopus (51) Google Scholar). Precursor Z may be assayed either by the appearance of fluorescent compound Z following oxidation of a sample (9Johnson J.L. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1989; 264: 13440-13447Abstract Full Text PDF PubMed Google Scholar, 14Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 110-116Crossref PubMed Google Scholar) or by the production of active molybdopterin upon incubation with a source of molybdopterin synthase(11Pitterle D.M. Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13506-13509Abstract Full Text PDF PubMed Google Scholar, 12Pitterle D.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13499-13505Abstract Full Text PDF PubMed Google Scholar, 15Johnson J.L. Wuebbens M.M. Mandell R. Shih V.E. J. Clin. Invest. 1989; 83: 897-903Crossref PubMed Scopus (51) Google Scholar).To date, no information is available regarding the initial steps of cofactor biosynthesis in any organism. However, the presence of molybdopterin as the essential component of all molybdenum cofactors raised the possibility that the early steps in the biosynthetic pathway of the molybdenum cofactor could be similar or identical to those of other pteridine biosynthetic pathways. Currently, three major routes for the synthesis of pteridines have been identified, leading to the formation of folates and riboflavin in plants and microorganisms and the synthesis of tetrahydrobiopterin (H4B) 1The abbreviations used are: H4BtetrahydrobiopterinH2NTP7,8-dihydroneopterin triphosphateARAPP2,5-diamino-6-ribitylaminopyrimidine 5′-phosphatept-6-COOHpterin-6-carboxylic acidHPLChigh performance liquid chromatography. and other nonconjugated pterins in animals. In all three pathways, GTP serves as the initial precursor for the pterin or pteridine rings of these molecules as shown through extensive studies involving the incorporation of radioactively labeled compounds into pterins and flavins as well as their biosynthetic intermediates by both whole cells and cell-free extracts. Taken together, these experiments demonstrated that in the three pathways, all of the carbons and nitrogens of guanine with the exception of the C-8 carbon are retained during the synthesis of the bicyclic ring structures of pteridines(16Levy C.C. J. Biol. Chem. 1964; 239: 560-566Abstract Full Text PDF PubMed Google Scholar, 17Reynolds J.J. Brown G.M. J. Biol. Chem. 1964; 239: 317-325Abstract Full Text PDF PubMed Google Scholar, 18Watt W.B. J. Biol. Chem. 1967; 242: 565-572Abstract Full Text PDF PubMed Google Scholar, 19McNutt W.S. J. Biol. Chem. 1954; 210: 511-519Abstract Full Text PDF PubMed Google Scholar, 20McNutt Jr., W.S. J. Biol. Chem. 1956; 219: 365-373Abstract Full Text PDF PubMed Google Scholar, 21McNutt W.S. J. Am. Chem. Soc. 1961; 83: 2303-2307Crossref Scopus (10) Google Scholar, 22Brown G.M. Blakley R.L. Benkovic S.J. Folates and Pterins. Vol. 2. John Wiley & Sons, New York1985: 115-154Google Scholar). These findings were clarified by the purification and characterization of the enzyme GTP cyclohydrolase I, which converts GTP to the reduced pterin, H2NTP(23Burg A.W. Brown G.M. J. Biol. Chem. 1968; 243: 2349-2358Abstract Full Text PDF PubMed Google Scholar, 24Yim J.J. Brown G.M. J. Biol. Chem. 1976; 251: 5087-5094Abstract Full Text PDF PubMed Google Scholar). In this reaction, as shown in Fig. 2, the C-8 guanine carbon is eliminated as formate, and the carbons of the ribose ring are utilized to generate the six-membered pyrazine ring of the pterin ring system(22Brown G.M. Blakley R.L. Benkovic S.J. Folates and Pterins. Vol. 2. John Wiley & Sons, New York1985: 115-154Google Scholar). Carbons 1′ and 2′ of the ribose are incorporated into the pterin ring, while the 3′, 4′, and 5′ carbons become the 1′, 2′, and 3′ carbons, respectively, of the six-alkyl side chain of H2NTP, which then serves as the common biosynthetic intermediate for both the folates and H4B.Figure 2:Initial steps in the known pathways of pteridine biosynthesis. Top, conversion of GTP to H2NTP as catalyzed by the enzyme GTP cyclohydrolase I. During this concerted reaction, all intermediates are protein-bound. Bottom, conversion of GTP to ARAPP by the enzyme GTP cyclohydrolase II.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In plants and microorganisms, a pteridine is also formed as an intermediate during the synthesis of the isoalloxazine ring system of riboflavin. The purification and characterization of a second, distinct E. coli cyclohydrolase (GTP cyclohydrolase II) which converts GTP to a phosphorylated ribitylaminopyrimidine revealed the nature of the initial reaction in this pathway(25Foor F. Brown G.M. J. Biol. Chem. 1975; 250: 3545-3551Abstract Full Text PDF PubMed Google Scholar). Again, the initial step is the loss of the C-8 carbon of a molecule of GTP as formate. However, the reaction catalyzed by GTP cyclohydrolase II results in the production of ARAPP as shown at the bottom of Fig. 2(25Foor F. Brown G.M. J. Biol. Chem. 1975; 250: 3545-3551Abstract Full Text PDF PubMed Google Scholar). Although all of the original guanosine carbons and nitrogens with the exception of C-8 are again incorporated into the final structure, the ribose carbons of GTP are not incorporated into the pteridine ring system, but are retained in toto as the ribityl group of riboflavin.In light of these studies, it was reasonable to investigate whether a guanine derivative also serves as the in vivo precursor of molybdopterin, with compounds such as H2NTP or ARAPP serving as common intermediates in the molybdopterin and folate or riboflavin pathways. Previous attempts at verification of these possibilities by directly labeling molybdopterin or its stable derivatives in a variety of systems had proven unsuccessful. These experiments were hampered by the extreme lability of molybdopterin as well as its relatively low abundance in wild type cells compared to the folates and flavins. However, the discovery and structural characterization of precursor Z and its oxidation product, compound Z, greatly increased the feasibility of in vivo labeling experiments in whole cells. In particular, precursor Z accumulates in the E. coli mutants moeB and moaE in amounts much higher than the molybdopterin content of wild type cells, and compound Z is stable and easily purified. In addition, since both compound Z and precursor Z contain all 10 of the carbon atoms present in molybdopterin, any information derived from in vivo labeling of carbons in compound Z would be immediately relevant to the elucidation of the early steps in molybdopterin biosynthesis.The experiments delineated in this report detail the results obtained from the analysis of compound Z purified from moeB cells cultured on minimal media supplemented with variously radiolabeled forms of guanosine. A method of release and purification of compound Z from these cells as well as from the culture medium is described, and a procedure for the analysis of the presence of label in both the side chain and pterin ring carbons, accomplished by degradation of the compound Z to pt-6-COOH and free pterin, is also delineated. The results obtained from these experiments indicate that while a guanosine derivative is indeed the initial in vivo precursor of molybdopterin biosynthesis in E. coli, the pathway by which the guanosine derivative is converted to molybdopterin is unlike all previously known pterin biosynthetic pathways.MATERIALS AND METHODSChemicals and ReagentsAmmonium chloride, sodium chloride, KH2PO4, Na2HPO4, and glucose were from Mallinckrodt. CaCl2 and KMnO4 were from J.T. Baker Chemical Co. Thiamine hydrochloride, QAE-Sephadex, and ribose were from Sigma. MgSO4, NaIO4, Florisil, and HPLC-grade ammonium acetate, methanol, and acetone were from Fisher. Chicken intestine alkaline phosphatase was from Worthington, and Cytoscint ES∗ scintillation mixture was from ICN. [U-14C]Guanosine in 10% ethanol with a specific radioactivity of 500 mCi/mmol was from Research Products International. [8-14C]Guanosine (56 mCi/mmol) and [8-3H]guanosine (15 Ci/mmol) in 2% ethanol were from Moravek Biochemicals. [8,5′-3H]GDP in 50% ethanol (10 Ci/mmol) was from DuPont NEN. All radiolabeled compounds had a radioactive purity ≥98%.Dephosporylation of [8,5′-3H]GDPAfter evaporation to dryness in air, the GDP was resuspended in 250 μl of H2O followed by the addition of 25 μl of 0.5 M MgCl2 and 20 μl of alkaline phosphatase (2 mg/ml of H2O). After overnight incubation, the alkaline phosphatase was inactivated by heating the solution in a boiling H2O bath for 45 s.Growth of moeB CellsLabeling of E. coli moeB cells was performed in 2.8-liter Fernbach flasks shaken vigorously at 37°C. Cells were inoculated into 1 liter of M-9 medium (0.5 g of NaCl, 1.0 g of NH4Cl, 3.0 g of KH2PO4, and 6.0 g of Na2HPO4/liter) supplemented with 15 ml of 20% glucose, 5 ml of 0.1 M CaCl2, 1 ml of 1.0 M MgSO4, 1 ml of 2 mg/ml thiamine, and 1 ml of a 1:10 dilution of Vogel Medium N stock trace element solution(26Davis R.H. De Serres F.J. Methods Enzymol. 1970; 17a: 79-143Crossref Scopus (932) Google Scholar). For growth on ribose as well as glucose, 5 ml of a 20% solution of this sugar was also added. Radioactive guanosine (54 μCi of U-14C, 25 μCi of 8-14C, 60 μCi of 8-3H, or 75 μCi of 8,5′-3H) was added immediately after inoculation, and the cells were cultured for approximately 34 h.Purification of Compound ZThe combined medium and cells from a 1-liter culture were acidified to pH 1.8 with concentrated HCl, followed by oxidation with 6 ml of 1% I2, 2% KI in H2O to convert all precursor Z to compound Z. After 20 min, the pH was adjusted to 8-9 by the addition of solid NaOH, and the cell debris was pelleted by centrifugation at 5,000 rpm in a Sorvall RC-3B centrifuge for 20 min. The supernatant was applied to a 2.5 × 10.0-cm column of QAE-Sephadex (acetate form) in three batches. After washing with 150 ml of H2O and 350 ml of 0.01 N acetic acid, compound Z was eluted from each column with 0.01 N HCl. The fractions comprising the third, and last, blue fluorescent band from each column were pooled and applied to individual 2.5 × 10.0-cm columns of Florisil resin. After washing the column with 50 ml of 0.01 N HCl and eluting with 22.5% acetone in H2O, the fluorescent fractions from the three batches were combined and rotoevaporated to dryness before final purification by reverse phase HPLC. The remaining fluorescent fractions from QAE-Sephadex chromatography could be used for the isolation of pterin and pt-6-COOH as described later.HPLC purification employed successive chromatography on an Alltech 10-μm C-18 column (4.6 × 250 mm) equilibrated in 2% methanol, pH 2, 0.5% methanol, pH 2, 0.01 N HCl, and 1 mM ammonium acetate, pH 5. After concentration to dryness and resuspension in 50 mM ammonium acetate, pH 5, the absorption spectrum of the compound Z was recorded and the concentration determined based on a molar extinction of 16,785 at 310 nm. Aliquots were then removed for quantitation of label using a Beckman LS 1801 scintillation counter. All HPLC analyses were performed at room temperature and utilized a Hewlett-Packard 1090 solvent delivery system. Eluting material was monitored for absorbance with a Hewlett-Packard 1040A diode array detector and for fluorescence with a Hewlett-Packard 1046A programmable fluorescence detector.Formation of pt-6-COOH from Compound ZThe remainder of the compound Z sample was adjusted to pH 12-13 with 1 N NaOH. An excess of 25 mM KMnO4 in H2O was added, and the sample was placed in a boiling water bath. Additional permanganate was added during the oxidation to maintain a bright purple color. After 20 min, the excess KMnO4 was reduced by the addition of 100 μl of 95% ethanol, and the precipitated MnO2 was removed by passing the sample through a 0.22-μm Costar Spin-X filter in an Eppendorf microcentrifuge. The original sample tube and the filter were then washed with 0.5 ml of 1 N NH4OH which was added to the filtrate. The entire sample was neutralized with 4 N HCl and concentrated to approximately 0.7 ml by rotoevaporation prior to injection onto a C-18 HPLC column equilibrated in 50 mM ammonium acetate, pH 5. The pt-6-COOH peak was collected directly as it emerged from the UV detector (10Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13493-13498Abstract Full Text PDF PubMed Google Scholar) and spectrally quantitated based on a molar extinction coefficient at 344 nm of 7410(27Pfleiderer W. Blakley R.L. Benkovic S.J. Folates and Pterins. Vol. 2. John Wiley & Sons, New York1985: 43-114Google Scholar). Aliquots were then removed for liquid scintillation counting and determination of specific radioactivity.Formation of Pterin from pt-6-COOHThe remaining pt-6-COOH was transferred to a 10-ml Pyrex beaker which was placed under an inverted, long-wavelength UV transilluminator (Ultra-Violet Products) with the sample 4-5 cm from the light source. The beaker was illuminated continuously for 15 h with the addition of aliquots of H2O to maintain a sample volume just sufficient to cover the bottom of the beaker. The entire sample was then injected onto a C-18 HPLC column equilibrated in 50 mM ammonium acetate, pH 5, containing 0.5% methanol. The pterin peak was collected and spectrally quantitated based on a molar extinction coefficient at 339 nm of 6170(27Pfleiderer W. Blakley R.L. Benkovic S.J. Folates and Pterins. Vol. 2. John Wiley & Sons, New York1985: 43-114Google Scholar).Isolation of Pterin and Bulk pt-6-COOH from Cells and Media Labeled with [8-14C]GuanosinePterin and pt-6-COOH were purified directly from the appropriate QAE-Sephadex fractions. For purification of pterin, the fractions containing the first blue fluorescent band were pooled and applied to a Florisil column similar to those used for compound Z purification. After washing with 0.01 N HCl and 200 ml of 22.5% acetone, pterin was eluted with a mixture of four volumes of 22.5% acetone and one volume of 1 N NH4OH. The pterin-containing Florisil fractions were pooled and concentrated to dryness by rotoevaporation. After resuspension in 1 ml of 0.01 N NaOH, final purification was achieved by two sequential injections of the entire sample onto a C-18 HPLC column equilibrated in 50 mM ammonium acetate, pH 5, with 0.5% methanol. Using this procedure, the total yield of pterin from 1 liter of cells and conditioned medium was approximately 5 μg.To obtain pt-6-COOH, the middle fluorescent QAE-Sephadex band and the 22.5% acetone wash obtained from purification of pterin on Florisil were combined and permanganate oxidized in order to convert all pterins and folates to pt-6-COOH. After ethanol oxidation of excess KMnO4, the entire sample was filtered through two layers of Whatman paper on a Buchner funnel to remove solid MnO2. The sample was concentrated to dryness by rotoevaporation, and the pt-6-COOH purified by chromatography on a C-18 HPLC column equilibrated in 50 mM ammonium acetate, pH 5. The total yield of pt-6-COOH was approximately 25 μg.RESULTSPurification of Compound ZAlthough the E. coli molybdopterin-deficient mutants moeB and moaE produce comparable amounts of precursor Z, the moeB mutant was arbitrarily chosen as the source of compound Z for all labeling experiments described here(9Johnson J.L. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1989; 264: 13440-13447Abstract Full Text PDF PubMed Google Scholar). Initial attempts to purify compound Z from moeB cells cultured on unlabeled, minimal media yielded only 10-15% of the amount of compound Z obtained from moeB cells cultured on rich media(9Johnson J.L. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1989; 264: 13440-13447Abstract Full Text PDF PubMed Google Scholar). Le Van et al. (28Le Van Q. Keller P.J. Bown D.H. Floss H.G. Bacher A. J. Bacteriol. 1985; 162: 1280-1284Crossref PubMed Google Scholar) and Bacher et al. (29Bacher A. Le Van Q. Keller P.J. Floss H.G. J. Biol. Chem. 1983; 258: 13431-13437Abstract Full Text PDF PubMed Google Scholar, 30Bacher A. Le Van Q. Keller P.J. Floss H.G. J. Am. Chem. Soc. 1985; 107: 6380-6385Crossref Scopus (20) Google Scholar) have utilized in vivo 13C labeling of riboflavin and its intermediates to study flavin biosynthesis in a number of organisms. In the course of these experiments, the labeled products were purified from the culture media rather than from the bacterial cells. This strategy yielded milligram quantities of labeled products from relatively small volumes of culture media. In view of these results, the possibility of purifying compound Z from the moeB culture media was examined.A preliminary experiment indicated that significant levels of compound Z could be isolated from the culture medium of moeB cells. Accordingly, a method for purification of compound Z from the entire cell culture was developed as described under “Materials and Methods.” This procedure yielded approximately 100 μg of compound Z from a 1-liter culture, a 30-fold increase over the amount purified from the cells alone.Sequential Cleavage of Compound ZIn order to assess the distribution of label between the ring and side chain positions of compound Z, a method involving sequential cleavage of the side chain carbons of compound Z was developed as shown in Fig. 3. Oxidation of compound Z with excess potassium permanganate under alkaline conditions at 100°C resulted in the loss of the three terminal side chain carbons. The product of this reaction, pt-6-COOH (31Forrest H.S. Mitchell H.K. J. Am. Chem. Soc. 1954; 76: 5658-5662Crossref Scopus (11) Google Scholar, 32Johnson J.L. Hainline B.E. Rajagopalan K.V. J. Biol. Chem. 1980; 255: 1783-1786Abstract Full Text PDF PubMed Google Scholar) is stable, highly fluorescent, and easily separated from compound Z by reverse phase HPLC. Extended illumination of the pt-6-COOH with UV light was then employed for cleavage of the remaining side chain carboxylate carbon as CO2 to yield free pterin (33Lowry O.H. Bessey O.A. Crawford E.J. J. Biol. Chem. 1949; 180: 389-398Abstract Full Text PDF PubMed Google Scholar), which could be purified by HPLC. A comparison of the specific radioactivities of the two resulting pterin derivatives with that of the original compound Z was used to evaluate labeling of the original side chain carbons.Figure 3:Degradation scheme for compound Z resulting in sequential cleavage of the side chain carbons to produce free pterin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Labeling with [U-14C]GuanosineDue to the poor transport of phosphorylated molecules into bacterial cells, labeled guanosine rather than any of the nucleotides was used. [U-14C]Guanosine was the first choice for in vivo labeling since it offered the greatest possibility of transfer of labeled carbons to precursor Z. Table 1 shows the distribution of label in compound Z purified from media supplemented with 54 μCi of [U-14C]guanosine/liter. For each of four separate cultures, the specific radioactivities of the purified compound Z, pt-6-COOH, and pterin, as well as the percentage of label lost after each individual cleavage are listed. The presence of label in compound Z in every case indicated the incorporation of carbon atoms from guanosine into precursor Z during molybdopterin biosynthesis. Alkaline permanganate cleavage of the three terminal side chain carbons from compound Z resulted in an average loss of 13% of the original specific radioactivity, demonstrating that one or more of these three carbons originated from guanosine. A further loss of 14C label upon UV treatment of the pt-6-COOH indicated that the C-1′ carbon of precursor Z had also been labeled by [U-14C]guanosine. These results demonstrate that a guanine or guanosine derivative is the initial precursor for molybdopterin biosynthesis.Tabled 1To determine whether substantial cleavage of the ribose portion of the guanosine was occurring prior to incorporation into precursor Z, labeling of moeB cells with [U-14C]guanosine was performed in minimal media supplemented with ribose as well as glucose. As seen in the last two sets of data in Table 1, although the addition of cold ribose to the growth medium did decrease the specific radioactivity of the compound Z purified from these cultures, it did not affect the overall distribution of label in that compound Z. Hence, cleavage of the guanosine by endogenous nucleosidases prior to incorporation into precursor Z did not appear to be a contributing factor to the observed pattern of carbon transfers.Labeling with [8-14C]GuanosineThe results of in vivo labeling with [U-14C]guanosine suggested that the initial step in molybdopterin biosynthesis in E. coli could be catalyzed by a GTP cyclohydrolase or a similar enzyme. To test this possibility, moeB cells were cultured in minimal media supplemented with [8-14C]guanosine. If molybdopterin biosynthesis does indeed proceed initially through the action of a cyclohydrolase I- or II-type reaction, then little, if any, label would be expected to be transferred from the C-8 position of guanosine to precursor Z. The results are shown in Table 2. Surprisingly, the isolated compound Z was labeled by the" @default.
• W2019965297 created "2016-06-24" @default.
• W2019965297 creator A5020639714 @default.
• W2019965297 creator A5080412705 @default.
• W2019965297 date "1995-01-01" @default.
• W2019965297 modified "2023-09-29" @default.
• W2019965297 title "Investigation of the Early Steps of Molybdopterin Biosynthesis in Escherichia coli through the Use of in Vivo Labeling Studies" @default.
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