The Biosynthesis of Folic Acid

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 242, No. 18, Issue of September 25, pp , 1967 Printed in II.S.A. The Biosynthesis of Folic Acid VII. ENZYMATIC SYNTHESIS OF PTERIDINES FROM GUANOSINE TRIPHOSPHATE* THEODORE H. D. JONEST AND GENE M. BROWN (Received for publication, April 24, 1967) From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 2139 SUMMARY Enzyme preparations from Escherichia co2i catalyze the conversion of various species cf Z-amino-4-hydroxy-6-(polyhydroxyalkyljdihydropteridines to dihydropteroic acid. Of these compounds, the one utilized most efficiently by the enzyme system is 2-amino-4-hydroxy-6-(D-erythro-1,2,3 - trihydroxypropyl)dihydrcpteridine. Evidence is presented to show that the latter campaund is converted by a heat-stable enzyme or enzymes to 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine, a compound which is used directly as substrate for the formation cf dihydropteroate and thus represents an intermediate in the conversion of the polyhydroxyalkyldihydropteridines to dihydropteroate. Evidence, is presented which indicates that the enzyme system that catalyzes the synthesis of dihydropteroate from p-aminobenzoate and guanosine triphosphate will convert GTP to pteridine intermediates when p-aminobenzoate and ATP are left out cf the reaction mixture. The pteridines that have been identified as praducts cf this transformation are 2 - amino hydroxy hydroxymethyldihydropteridine and 2 -amino hydroxy (erythro - 1,2,3 - trihydroxy - propyl)dihydropteridine. Smaller quantities of the corresponding ihreo isomer of the latter compound are also made enzymatically from GTP, but the significance of the formation of this compound is not cleer. Previous investigat ions from this laboratory (2, 3) and other laboratories (4-6) have established that hydroxymethyldihydropteridinel can be utilized efficiently for the enzymatic synthesis * These investigations were supported by Grant GB-2153 from the National Science Foundation and by Grant AM 3442 from the United States Public Health Service. For Paper VI of this series see Reference 1. $ A Karl T. Compton Predoctoral Fellow of the Nutrition Foundation. Present address, Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts. 1 In this paper the term pteridine will refer to 2.amino-4-hydroxypteridine. Various substituents on position 6 of the pteri- of either dihydropteroic acid (with p-aminobenzoate as cosub- &rate) or dihydrofolic acid (with p-aminobenzoylglutamic acid as cosubstrate). Reynolds and Brown (7, 8) later showed that guanosine or guanine nucleotides can replace the pteridine requirement in this system, and they presented evidence which led to the conclusion that the guanine compounds are converted enzymatically to the pteridine moiety of folic acid. This conclusion has now been confirmed by other investigators (9, 1). Reynolds and Brown (8) suggested that this transformation occurs by a biosynthetic pathway, similar to that originally formulated by other investigators (II, 12), which includes dihydroneopterin and hydroxymethyldihydropteridine as intermediates. Evidence that dihydroneopterin might be an intermediate was obtained with the observation that this compound can be utilized in place of hydroxymethyldihydropteridine as substrate for the synthesis of folate compounds (13, 14). Although the investigations summarized above strongly suggest that dihydroneopterin and hydroxymethyldihydropteridine are intermediates in the enzymatic conversion of guanosine and guanine nucleotides to folic acid, no direct evidence in support of this suggestion has yet been presented. The work described in this paper shows that an enzyme system from Es&e&h& coli catalyzes the synthesis of both of these postulated intermediates from guanosine triphosphate. EXPERIMENTAL PROCEDURE Materials-Folic acid, sodium ascorbate, D-ribose, L-xylose, and L-arabinose were purchased from Calbiochem; Tris, D- dine will be indicated when referred to in the text. Thus, 2- amino-4-hydroxy-6-hydroxymethylpteridine will be referred to simply as hydroxymethylpteridine. Neopterin is the trivial name that will be used for 2-amino-4-hydroxy-6-trihydroxypropylpteridine. The various nossible isomers of this compound due to differences in configuration in the trihydroxypropyl side chain will be distinguished by the correct prefix; i.e. the erythro isomers become n-erythro-neopterin and L-erythro-neopterin, and the three isomers become n-three-neopterin and L-three-neopterin. Occasionally, reference will be made to the pteridines containing the side chain in position 7. To distinguish these from the compounds with side chains on position 6, a 7 will be used as a prefix, i.e. 7- n-erythro-neopterin. 7,8-Dihydropteridines will be referred to as dihydropteridines.

2 399 Biosynt.hesis of Folic Acid. VII Vol. 242, No. 18 OH FIG Amino-4-hydroxypteridine with various substituent.s on posit.ion 6. For example, hydroxymethylpteridine is the compound resulting when R = -CHzOH. n-erythro-neopterin OH OH results when I R = -C-C-CHzOH. I I HH I erythrose, and m-glyceraldehyde from Sigma; AB2 and n-xylose from Fisher Scientific Company; 2-mercaptoethanol, hydrazine, and acetaldehyde from Eastman Organic Chemicals; all nonradioactive nucleotides from P-L Biochemicals; bovine serum albumin from Pentex, Inc.; 2,4,5-triamino&hydroxypyrimidine sulfate from the Aldrich Chemical Company; phosphocellulose from Gallard-Schlesinger Chemical Manufacturing Corporation; GTP-UJ4C from Schwartz BioResearch; and ribose-l-14c from Nuclear-Chicago. Glucose-U-14C and purified alkaline phosphatase from E. coli were gifts from Drs. P. W. Robbins and A. M. Torriani, respectively. Dr. H. Rembold kindly provided samples of pure n-erythro-neopterin and 7-r-erythro-neopterin (see Fig. 1 for the formulas for these and related compounds). Pure biopterin was kindly provided by Drs. G. W. Kidder and V. Dewey. Microbiological Methods-Enzymatically produced dihydropteroic acid was determined by microbiological assay with Streptococcus faecalis 843 as described previously (2). For convenience, folic acid was used to prepare the standard curves in the assays; thus, results will be presented as folate equivalents produced, although dihydropteroic acid is known to be the product formed under the conditions used (2). Growth and preparation of extracts of E. coli B (a strain resistant to bacteriophage Tl and T5) were carried out as described in a previous paper (2). These crude extracts were treated with RNase and charcoal as described by Reynolds and Brown (8). Such preparations usually contained 15 to 2 mg per ml of protein. Protein was measured by the method of Lowry et al. (15) with bovine serum albumin as the standard. Preparation and PurQication of PteridinesHydroxymethylpteridine was prepared by the method described by Wailer et al. (16). Polyhydroxyalkylpteridines were synthesized as described by Rembold and Metzger (17). This method involves the condensation of 2,4,5-triamino-6-hydroxypyrimidine with the appropriate sugar. Pteridines synthesized in this manner and, in each case, the sugar used as reactant were as follows: D- erythro-neopterin from n-ribose; n-erythro-neopterin-7-4c from n-ribose-l-14c; n-erythro-neopterin from L-arabinose; D-threoneopterin from n-xylose; n-three-neopterin from n-xylose; r>-arabotetrahydroxybutylpteridine from n-glucose; and n-dihydroxyethylpteridine from n-erythrose. The crude reaction mixtures of all of these compounds contained numerous contaminating com- 2 The abbreviation used is: AB, p-aminobenzoic acid. pounds formed as by-products of the reaction. The desired compounds were obtained in pure form by subjecting the reaction mixtures to chromatography on phosphocellulose. The general procedures of Rembold and Metzger (17) were followed for preparing the phosphocellulose columns and for the operation of the columns. One useful modification of the published method was made in the preparation of the sample to be applied to the column. The solid material was dissolved in the minimum amount of normal NaOH, and the solution was adjusted to ph 7. with normal HCl. The copious brown precipitate which appeared was removed by filtration or by centrifugation. The filtrate or supernatant solution was pale yellow-green in color and contained little of the darker compounds which, if not removed, adsorb strongly to the phosphocellulose and interfere with the resolution achieved on the column. The fractionation of a crude preparation of n-erythro-neopterin on phosphocellulose resulted in the typical elution profile shown in Fig. 2. The first major component t,hat was eluted from the column (contained in Fractions 43 to 52) was identified as n-erythro-neopterin by its spectral characteristics (18), its behavior on paper chromatograms in four solvent systems (18), and the fact that oxidation by permanganate, as described by Rembold and Buschmann (19), yielded only 6-carboxypteridine (distinguished from 7- carboxypteridine by paper chromatographic methods (19)). The second major component eluted from the column (Fractions 53 to 64) was identified by its spectral and chromatographic characteristics (17) as 7-n-erythro-neopterin, which is a byproduct of the reaction. The third component (Fractions 64 to 83) has not been identified. The other pteridines were synthesized and purified in the same way, and elution profiles of these compounds from phosphocellulose columns were similar to the one shown in Fig. 2. Miscellaneous Methods-Optical densities and spectra were measured by a Perkin-Elmer 22 recording spectrophotometer. Published extinction coefficients were used to calculate the concentrations of n-erythro-neopterin (18), E = 2.51 X lo4 Mm FRACTION NUMBER FIG. 2. Purification of n-erythro-neopterin by chromatography on phosphocellulose. Crude n-erythro-neopterin (1 mg) was suspended in 4 ml of water and 1 N NaOH was added dropwise until all of the material was in solution. The solution was then adjusted to ph 7. with 1 N HCI. The resulting gelatinous, brown nrecioitate was removed bv filtration, The filtrate was applied to a phosphocellulose column (2 X 25 cm). After all of thematerial had entered the column, water was passed through at a flow rate of.5 ml per min. Fractions of 1 ml each were collected and ultraviolet light-absorbing materials were determined by measuring optical densities of the fractions at 272 rng.

3 Issue of September 25, 1967 T. H. D. Jones and G. M. Brown 3991 cm+ at 253 rnp in.1 N NaOH; and hydroxymethylpteridine 3, e = 2.2 x lo4 MFcrn- in.1 N NaOH. The extinction coefficients of the other polyhydroxyalkylpteridines were assumed to be equal to that of D-erythro-neopterin. Radioactivity in fractions from columns was measured with a Nuclear-Chicago 72 liquid scintillation counter. Radioactivity on paper chromatograms was located and the amounts estimated with a Packard 72 radiochromatogram scanner. Pteridines were reduced to dihydropteridines with sodium hydrosulfite as described by Friedkin, Crawford, and Misra (2). Reaction mixtures were prepared as described for each experiment to be presented, and incubation of the reaction mixture was carried out under anaerobic conditions as described previously (2). RESULTS Enzymatic Conversion of Polyhydroxyalkyldihydropteridines to Dihydropteroate-In a previous publication (13) we showed that each of the four diastereoisomers of dihydroneopterin can be utilized as substrate for the formation of dihydropteroate, catalyzed by enzymes present in a charcoal-treated extract of E. coli. The relative degrees of effectiveness of utilization of the dihydro forms of these compounds were found to be, in order of decreasing effectiveness: n-erythro-neopterin, L-threo-neopterin, and n-three-neopterin equal to L-erythro-neopterin. The fact that n-erythro-dihydroneopterin was used the most efficiently is an expected result since this compound is the one that would be predicted to be formed by the proposed biosynthetic TABLE I Abilities of certain dihydropteridines to be used as substrates for enzymatic synthesis of dihydropteroate Reaction mixtures contained, in a total volume of.14 ml: 2- mercaptoethanol,.7 M; Tris buffer (ph 8.6),.7 M; MgC12, 7 X 1OW M; ATP, 3.5 X 1e3 M; AB, 3.5 X 1m4 M; charcoal-treated enzyme preparation equivalent to.2 mg of protein; and dihydropteridine compound as indicated. Incubation was for 3; hours at 37 under anaerobic conditions. Production of dihydropteroate was measured as described in the Experimental Procedure. Dihydropteridine substrate added to reaction mixture Experiment A None... D-erythro-Dihydroneopterin... Dihydroxyethyldihydropteridine Dihydroxyethyldihydropteridine Experiment B None... o-erythro-dihydroneopterin.. D-arabo-Tetrahydroxybutyldihydropteridine... o-arabo-tetrahydroxybutyldihydropteridine... Experiment C None... n-ertyhro-dihydroneopterin.... Dihydrobiopterin Dihydrobiopterin Dihydrobiopterin SC Amount of dihydfoptendme ibstrate addet mpnoles Folate equivalents produced TABLE II Inhibition of enzymatic synthesis of dihydropteroate by %amino+hydroxydihydropteridine Reaction mixtures were prepared as described in Table I, except that the volume of the reaction mixtures was lo-fold greater, although concentrations of components were the same. Substrates and inhibitor were added in the amounts shown. Substrate added Experiment A n-erythro-dihydroneopterin.. n-erythro-dihydroneopterin.. D-erythro-Dihydroneopterin.. D-erythro-Dihydroneopterin.. Hydroxymethyldihydropteridine... Hydroxymethyldihydropteridine... Hydroxymethyldihydropteridine... Hydroxymethyldihydropteridine... Experiment B GMP GMP GMP GMP Amount of substrate added DihydFo- PtHldl$ mjmdes Folate :quivalents produced pathway from guanine nucleotides to folate (8). None of these compounds was utilized as well as hydroxymethyldihydropteridine. The results of experiments to test the abilities of other dihydropteridines to function as substrates are given in Table I. For each experiment the effectiveness of utilization of n-erythro-neopterin is also shown for purposes of comparison. Both dihydroxyethyldihydropteridine and n-arabo-tetrahydroxybutyldihydropteridine promoted the synthesis of dihydropteroate, although neither was utilized as substrate as well as n-erythro-dihydroneopterin. These results are similar to those reported by Mitsuda et al. (14), who used extracts of Bras&a pekinensis as a source of enzymes. Biopterin, 2-amino-4-hydroxy-6-(L-erythro-l,2 dihydroxypropyl)pteridine, is a naturally occurring compound which, in its reduced form, is known to function as a cofactor for various enzymatic hydroxylation reactions. The results of Table I show that the dihydro form of biopterin was not utilized. Considerations of the mechanisms by which polyhydroxyalkyldihydropteridines could be converted to the pteridine component of dihydropteroate suggest that these compounds are first converted to hydroxymethyldihydropteridine either by removal of all but 1 of the carbons of the side chain or by removal of all of the carbons of the side chain followed by addition of a l-carbon unit (probably formaldehyde) to position 6 of the dihydropteridine. The latter mechanism has been proposed (21) as the method of formation of hydroxymethyldihydropteridine. However, the operation of this pathway would mean that dihydropteridine (no substituent on position 6) would have to be an intermediate, and we have found that this compound not only is not utilized for enzymatic synthesis of dihydropteroate (in the presence or absence of a l-carbon compound, such as formaldehyde), but, as shown in Table II, is

4 Biosynthesis of Folic Acid. VII Vol. 242, No. 18 actually an inhibitor of the enzymatic synthesis of dihydropteroate from either guanine nucleotides, n-erythro-dihydroneopterin, or hydroxymethyldihydropteridine. Enzymatic Formation of Hydroxymethyldihydropteridine from D-erythro-Dihydroneopterin--The results described above clearly indicate that dihydropteridine is not an intermediate in the conversion of polyhydroxyalkyldihydropteridines to dihydropteroate, and therefore suggest that a compound such as D-erythrodihydroneopterin is converted to hydroxymethyldihydropteridine by removal of 2 carbons of the 3-carbon side chain. The fact that n-erythro-dihydroneopterin is utilized less effectively than hydroxymethyldihydropterine for the formation of dihydropteroate (13) suggests that in this process the conversion of werythrodihydroneopterin to hydroxymethyldihydropteridine may be the rate-limiting step. If this is true, it should be possible to devise a two-stage incubation procedure whereby one can detect the formation, during the first incubation period, of a compound (presumably hydroxymethyldihydropteridine) which, during the second incubation period, would be converted to dihydropteroate at a rate faster than n-erythro-dihydroneopterin would be converted to this product. The results of such an experiment, shown in Table III, indicate that such an intermediate product TABLE Detection of intermxliate in enzymatic conversion of n-erythro-dihydroneopterin to dihydropteroate During the first incubation period reaction mixtures were prepared to contain, in a total volume of.11 ml: 2-mercaptoethanol,.7 M; Tris (ph 8.6),.9 M; charcoal-treated extract equivalent to.8 mg of protein; and either n-erythro-dihydroneopterin (1 X 1-d M) or hydroxymethyldihydropteridine (2 X 1-E M). Reaction mixtures were incubated for 3 hours under anaerobic conditions at 37. The mixtures were then heated at 1 for 1 min, after which MgClz (1 pmole), AB (1 pmoles), ATP (.5 pmole), and charcoal-treated extract equivalent to.4 mg of protein were added to give a final volume of.16 ml. The reaction mixtures were then reincubated for l+ hours at 37 under anaerobic conditions. The formation of dihydropteroate was determined by microbiological assay. Reaction Components present during mixture first incubation period Enzyme,a HP Enzyme neopterin- III Component added after first, but prior to second, incubation period AB, Mg2+, ATP, en zyme AB, MgZ+, ATP, en zyme AB, Mg2+, ATP, enzyme AB, $I@+, ATP AB, Mg2+, ATP, enzyme AB, Mg2+, ATP, enzyme AB, Mg2+, ATP, enzyme Folate quivalents produced Yeopterin-Hz 12 Enzyme, neopterin-hz Enzyme, neopterin-hz (heated)b Enzyme, CHnOH-pteridine-Hza CI&OH-pteridine-Hz *2Pg a Enzyme refers to a charcoal-treated extract; neopterin-hz is an abbreviation for n-erythro-dihydroneopterin; and CH2Hpteridine-Hz stands for hydroxymethyldihydropteridine. 6 The reaction mixture was heated for 1 min at 1 after the addition of the components, and then incrtbated does accumulate during the first incubation period. Since the only components present in Reaction Mixture 1 (Table III) during the first incubation were n-erythro-dihydroneopterin and the enzyme preparation, the transformation of n-erythro-dihydroneopterin to the intermediate cannot be dependent on ATP, Mg2+, or any other cofactor. Prior to the second incubation, AB, ATP, Mg2+, and fresh enzyme preparation were added to allow the production of dihydropteroate. The difference between dihydropteroate synthesized in ReacCon Mixtures 1 and 3 (Table III) thus represents the amount produced as a result of the formation in the first incubation period (Reaction Mixture 1) of a compound that is converted to dihydropteroate at a rate faster than n-erythro-dihydroneopterin is converted to dihydropteroate during the second incubation period. It is also clear from the data presented in Table III that the enzyme preparation is needed during the second incubation (see Reaction Mixture 4), and that the enzyme or enzyme system that catalyzes the conversion of n-erythro-dihydroneopterin to the intermediate is stable to heating at 1 for 1 min (see Reaction Mixture 5). Reaction Mixtures 6 and 7 were included to show that no increase in the rate of formation of dihydropteroate results from prior incubation of hydroxymethyldihydropteridine with the enzyme preparation. Other experiments have indicated that the amount of the intermediate formed from n-erythro-dihydroneopterin, as measured by the two-stage incubation procedure described in Table III, is directly proportional to the amount of enzyme preparation present during the first incubation period. Thus, the procedure described in Table III can be used as an assay for the enzyme that converts n-erythro-dihydroneopterin to the intermediate. Also, we have shown that this enzyme activity is stable to heating at 1 for as long as 5 min, and that a 13.fold purification of this activity can be achieved by heating a charcoal-treated extract for 5 min followed by centrifugation to remove the resulting insoluble, inactive protein. A likely explanation for the observations described above is that hydroxymethyldihydropteridine is produced from D-erythrodihydroneopterin during the conversion of the latter compound to dihydropteroate. To test this possibility, n-erythro-dihydroneopterin-7-l% was incubated with an enzyme preparation, as described in Fig. 3. The incubated reaction mixture was passed through a phosphocellulose column from which all of the radioactivity was recovered as a broad peak. The fractions containing the radioactivity were combined, carrier hydroxymethylpteridine was added to the solution, and this material was subjected to chromatography on a second phosphocellulose column. The details of these procedures are given in Fig. 3. The failure to obtain separation of components on the first phosphocellulose column was probably due to the relatively high ionic strength of the incubated reaction mixture. The expectation that hydroxymethylpteridine would be the product present after passage of the material through the first column (even though hydroxymethyldihydropteridine is the expected enzymatic product) follows from our observations that dihydropteridines are easily oxidized during chromatography on phosphocellulose and on paper. It may be observed from Fig. 3 that a radioactive compound w-as eluted from the second column at exactly the same rate that carrier hydroxymethylpteridine was eluted (Fractions 7 through 9), i.e. the peak of radioactivity corresponds exactly to the optical density peak due to the carrier hydroxymethylpteridine. Radioactive material contained in

5 Issue of September 25, 1967 T. H. D. Jones and G. M. Brown 3993 Fractions 44 through 65 represents unreacted D-erythro-neopterin (formed by oxidation of the corresponding dihydro compound used to prepare the reaction mixture). Radioactive materials which were eluted earlier were unidentified nonenzymatic degradation products of n-erythro-neopterin. That the radioactive material which eluted in the same position as hydroxymethylpteridine was produced enzymatically was shown by the fact that no such product was detected when the experiment described in Fig. 3 was repeated in the absence of the enzyme preparation. Another enzymat.ic reaction mixture identical with that described in Fig. 3 was prepared and incubated for 18 hours, instead of 6; hours, in an attempt to increase the yield of product. The incubated reaction mixture was then subjected to the same chromatographic procedures described in Fig. 3, except that no carrier hydroxymethylpteridine was added to the material before application to the second phosphocellulose column. The pattern of radioactivity eluted from the second column resembled that shown in Fig. 3, except that larger amounts of degradation products were present, bti all of these were eluted from the column before either n-erythro-neopterin or hydroxymethylpteridine. The fractions containing the radioactive material which was eluted as a peak in the region where hydroxymethylpteridine should be eluted were combined, and the volume of the combined fractions was reduced to.5 ml by evaporation under reduced pressure. The material was subjected to paper chromatographic analysis as described in Table IV. The product behaved as hydroxymethylpteridine in the three solvent RADIOACTIVITY FRACTION NUMBER OPTICAL DENSITY i.4. I\ i \ FIG. 3. Elution pattern from phosphocellulose of hydroxymethylpteridine formed enzymatically from n-erythro-dihydroneopterin. A reaction mixture was prepared to contain in a total volume of 7.4 ml: 2-mercaptoethanol,.54 M; n-erythro-dihydroneopterin (193, cpm per pmole) 6.5 X 1+~ M; and charcoal treated extract equivalent to 93 mg of protein. Incubation was for 6$ hours at 37 under anaerobic conditions. The incubated reaction mixture was cooled to 4 and a 6.7-ml portion was applied to a phosphocellulose column (1 X 1.5 cm). Elution was carried out at 4 with water. Fractions (3. ml each) were collected at a rate of.18 ml per min. Determination of radioactivity present in the fractions showed that all of the radioactivity was eluted in a broad, unfractionated peak in Fractions 1 to 56. The contents of these fractions were combined,.9 pmole of hydroxymethylpteridine was added as carrier, and, after evaporation of the solution to approximately 2 ml, this material was applied to a second phosphocellulose column (1 X 1.7 cm). Elution was again carried out at 4 with water at a flow rate of.18 ml per min. Fractions of 4 ml each were collected and analyzed for optical density at 272 rnp and for radioactivity. The elution pattern from the second phosphocellulose column is shown. i TABLE IV Identification by paper chromatography of hydroxymethylpteridine as product formed from o-erythro-dihydroneopterin Standards (hydroxymethylpteridine and D-erythro-neopterin) were spotted on individual chromatogramn in lo--mole amounts. The amount of purified, enzymatic product (preparation as described in the text) that was spotted on each chromatogram was 4 mmoles. Development of the chromatograms was by the ascending method with the solvents shown. Zones of migration were located by observing blue fluorescence under ultraviolet light. Solvents that were used are as follows: Solvent 1, 3% NH&l; Solvent 2, 5yo bol;ic acid; and Solvent 3, isopropyl alcohol-5y boric acid (4: 1, by volume). Material Solvent1 1 Sol ent2~so* ent3 RF n-erythro-neopterin Hydroxymethylpteridine Purified, enzymatically formed product systems used. In addition, the developed chromatograms were subjected to analysis for radioactivity with a strip scanner. These analyses showed that a single radioactive zone was present after development with each of the three solvents, and in all three cases the radioactive zones were exactly coincident with the fluorescent zones. These results show that werythrodihydroneopterin can be converted enzymatically to a compound wh.ich, after being subjected to oxidative conditions during purification, can be identified as hydroxymethylpteridine. By other experiments not described here, we have shown that unreduced substrate (i.e. n-erythro-neopterin) cannot serve as substrate for this enzymatic transformation. Thus, it seems clear that the product formed directly from n-erythro-dihydroneopterin by the action of the enzyme is hydroxymethyldihydropteridine. Enzymatic Conversion of GTP to Dihydroneopterin and Hydroxymethyldihydropteridine-We have observed that the enzymatic formation of dihydropteroate is relatively less efficient from guanosine or guanine nucleotides than from n-erythro-dihydroneopterin or hydroxymethyldihydropteridine. Thus, it seems reasonable to expect that, in the conversion of guanine nucleotides to dihydropteroate, the rate-limiting reaction is concerned with the formation of dihydroneopterin from the guanine nucleotide. An experiment was designed which included a preliminary incubation period during which guanine nucleotide was incubated with enzyme preparation. This was followed by the addition to the reaction mixture of AB, ATP, and new enzyme preparation. The reaction mixture was then reincubated and the production of dihydropteroate was measured. The details of this experiment are given in Table V. The results show that GTP was converted to a product or products (during the first incubation period) which could be utilized more efficiently than GTP for the producation of dihydropteroate during the second incubation period (compare Reaction Mixtures 1 and 2, Table V). Other results presented in Table V show that neither GDP nor GMP can be utilized for the formation of these intermediates, an indication that these nucleotides are not direct precursors of folate compounds. Previously, these compounds, as well as guanosine, were shown to be converted to dihydropteroate (8) ; but ATP was present in the reaction mixtures and presumably they were

6 3994 Biosynthesis of Folic Acid. VII Vol. 242, No. 18 converted to the direct precursor, GTP, through the action of phosphate-transferring enzymes. Other experiments have indicated that magnesium ions are not needed in the production of intermediates from GTP, and that the ability of such intermediates to serve as precursors of dihydropteroate is reduced by approximately 6% when preparations of these materials are stored for 24 hours at, exposed to air. Most of the dihydropteroate-precursor activity could be recovered by treatment with sodium hydrosulfite, a fact which suggests that the intermediate, or intermediates, was capable of being reversibly inactivated by oxidation. The observations described above suggested that GTP is converted enzymatically to one or more dihydropteridine compounds which are intermediates in the pathway for the synthesis of dihydropteroate from GTP. In order to test this possibility, experiments were performed in which GTP-U-W was incubated with an enzyme preparation, and the incubated reaction mixture was then subjected to chromatographic procedures on phosphocellulose by which pteridines can be separated from GTP. This experiment is described in Fig. 4. This figure shows that two major radioactive peaks were obtained from the phosphocellulose column. The second peak (Peak 2) corresponded exactly with the elution pattern of the carrier hydroxymethylpteridine, shown in the figure by measurements of optical densities at 272 mp. The other major radioactive peak (Peulc 1) appeared in the general position to be expect,ed for neopterin. Materials from Peaks 1 and 2 were subjected to paper chromatography as described in Table VI. The results show that material from Peak 1 migrated as n-erythro-neopterin in three different solvent systems, and that material from Peak 2 migrated as hydroxymethylpteridine in the same three solvents. The fractions (52 through 75) constituting Peak 2, which contained hydroxymethylpteridine, were combined and concentrated TABLE Formation of intermediates in conversion of GTP to dihydropteroate Reaction mixtures were prepared to contain, per.13 ml: 2- mercaptoethanol,.78 M; Tris buffer (ph 8.6),.78 M; MgC12, 7.8 X 1-S M; Na2HP4,.16 M; charcoal-treated extract equivalent to 1.2 mg of protein; and guanine nucleotide, 3 X lo-4 M. The reaction mixtures were incubated at 37 for 2 hours under anaerobic conditions. The mixtures were then heated for 1 min at loo", cooled, and supplemented with AB (1 pmoles), ATP (.5 pmole), and charcoal-treated extract (equivalent to.6 mg of protein). The reaction mixtures (total volume for each was.18 ml) were reincubated at 37 for 2 hours under anaerobic conditions. Reaction Nucleotide present during first mixture incubation V Components added after first incubation and prior to second incubation GTP Complete system GTP, omit enzyme Complete system GDP Complete system GDP, omit enzyme Complete system GMP Complete system GMP, omit enzyme Complete system a The complete system includes AB, ATP, and charcoal-treated extract. Folate equivalents d%% ml.@ $ 5 3 Q E 25 5 a 2 $ 15 c 2 1 G 2 5 \ RADIOACTIVITY PEAK I \ PEAK 2 % : \ P FRACTION NUMBER FIG. 4. Elution pattern from phosphocellulose of compounds formed enzymatically from GTP-U-W. A reaction mixture was prepared to contain, in each 6.6 ml of reaction volume: 2-mercaptoethanol,.6 M; Tris buffer (ph 8.6),.6 M; NalHP4, 6 X 1c3 M; GTP-U-l%, 1.8 X 1m4 M (8 X lo6 cpm); and charcoal-treated extract equivalent to 96 mg of protein. The reaction mixture was incubated anaerobically for 36 hours at 37. MgCL (1 rmoles) and alkaline phosphatase, 2,ug of protein, were then added and the reaction mixture was reincubated for 1 hour at 37 to dephosphorylate any phosphorylated pteridines that may have been formed, since subsequent chromatography on phosphocellulose would not have separated phosphorylated compounds. After the second incubation, the reaction mixture was heated at 1 for 1 min and the resulting precipitated protein was removed by centrifugation. The supernatant solution was applied to a phosphocellulose column (1. X 11.2 cm). Water was then passed through the column at room temperature and 3-ml fractions were collected at a rate of.15 ml per min. A broad radioactive peak of material (Fractions 15 to 48) was obtained which also possessed ability to be converted enzymatically to dihydropteroate (after reduction with sodium hydrosulfite). These fractions were combined and evaporated under reduced pressure to 1 ml. Carrier hydroxymethylpteridine (.64 pmole) was added and the solution was applied to a second phosphocellulose column (1. X 1.2 cm). Water was then passed through the column at room temperature and fractions of 3. ml were collected at a rate of.15 ml per min. Radioactivity and optical density at 272 mp for each fraction were determined. The elution pattern from the second phosphocellulose column is shown..9 b.6 % -I.5 ;.4 z 5.3 : ; F TABLE VI Paper chromatographic properties of products formed enzymatically from GTP Amounts of material from Peaks 1 and 3, Fig. 4, equal to 15 to 2 cpm from each peak, were spotted individually on paper, along with authentic hydroxymethylpteridine and D-erythroneopterin. The amount of each of these standards spotted on each chromatogram was 1 mpmoles. Solvents used were: Solvent 1, 3% ammonium chloride; Solvent 2, 4% trisodium citrate; and Solvent 3,5/o boric acid. Development of the chromatograms at room temperature was by the ascending technique. Radioactive zones of migration were located by use of a strip scanner. Material spotted Hydroxymethylpteridine n-erythro-neopterin Material from Peak Material from Peak

7 Issue of September 25, 1967 T. H. D. Jones and G. M. Brown 3995 by evaporation to approximately 7 ml, and the white precipitate of hydroxymethylpteridine that was obtained was collected by centrifugation and recrystallized five times from water. The specific radioactivity for each crop of crystals was measured and found to remain constant (168 cpm per mpmole) for the five recrystallizations. The recrystallized material was identified as hydroxymethylpteridine by its absorption spectrum and its RF values in the solvents listed in Table VI. These experiments indicate that one product produced enzymatically from GTP is hydroxymethyldihydropteridine. The latter compound is labile to oxidation during isolation, which accounts for the fact that the compound isolated is in the oxidized state. Fractions 3 to 5 (Peak 1, Fig. 4) were combined and, after the addition of carrier n-erythro-neopterin, the material was subjected to chromatography on phosphocellulose (see Fig. 5 for experimental details). The results given in Fig. 5 show that the major radioactive peak did not correspond exactly to the optical density peak exhibited by the authentic n-erythro-neopterin added as carrier. In order to discover the reason for this discrepancy, material from the leading edge of the radioactive peak (Fractions 38 through 4) and material from the trailing edge (Fractions 47 through 53) were each subjected to rechromatography on individual columns of phosphocellulose, as described in Fig. 6. The results show that radioactive material from the leading edge was eluted from the phosphocellulose column at exactly the same place as the carrier n-erythro-neopterin, located by measuring optical densities at 272 rnp (Fig. 64). On the other hand, material taken from the trailing edge of the peak shown in Fig. 5, after rechromatography, yielded a radioactive peak that was not coincident with the carrier n-erythro-neopterin (Fig. 6B). These results indicate that theradioactivepeak shown in Fig. 5 contained at least two components, one of which is probably o-erythro-neopterin and the other anunidentified compound that is eluted slightly later. Experiments designed to reveal elution patterns of the other isomers of neopterin have shown that n-erythro-neopterin and L-erythro-neopterin are eluted from RADIOACTIVITY FRACTION NUMBER FIG. 5. Chromatography on phosphocellulose of material contained in Fractions 3 to 6, Fig. 4. Fractions 3 to 6 (Fig. 4) were combined and reduced in volume to 2 ml by evaporation under reduced pressure. Approximately 1 mg of carrier D-erythroneopterin was added and the material applied to a phosphocellulose column (2.2 cm X 16.5 cm). Water was then passed through the column at room temperature and lo-ml fractions were collected at a rate of.4 ml per min. The radioactivity and optical density of each fraction were determined. z I I = FE % $ 4. l? 48 I RADIOACTIVITY..,! ; I? I FRACTION NUMBER IO FIG. 6. Rechromatography on phosphocellulose of Fractions 38 to 4 (A) and Fractions 47 to 53 (B) from the phosphocellulose column described in Fig. 5. Fractions 38 to 4 were combined into one fraction and Fractions 47 to 53 were combined in another. Each combined fraction was then reduced in volume to 2 ml by evaporation under reduced pressure. Each solution was subjected to chromatography on individual phosphocellulose columns. The sizes of the columns were 1 X 23 cm for Fractions 38 to 4 (A) and 1 x 24.5 cm for Fractions 47 to 63 (B). Each column was developed with water at room temperature. Fractions of 3. ml were collected at a rate of.2 ml per min. Optical density at 272 rnp and radioactivity of each fraction were determined. TABLE VII Identifcation of threo-neopterin as product formed enzymatically from GTP An enzymatic reaction mixture was prepared (containing GTP- U-l%) and incubated as described in Fig. 4. A nonradioactive carrier amount of L-threo-neopterin (.6 pmole) was added to the incubated react,ion mixture, and threo and erythro isomers of neopterin were separated on phosphocellulose columns as described in Figs. 5 and 6. The slower eluting of the two radioactive components was collected and concentrated by evaporation. This material also contained the carrier n-threo-neopterin as judged by optical density measurements. An amount of this material equal to approximately 1 cpm was spotted on Whatman No. 1 paper along with standards of synthetic D-erythroneopterin, n-threo-neopterin, and n-threo-neopterin (approximately 1 mpmoles each). The chromatogram was developed by the descending technique with isopropyl alcohol-by boric acid (by volume) as solvent. To improve the resolution, the solvent front was allowed to run off the chromatogram. Time of development was for 36 hours at room temperature. Zones of migration were determined by observing blue fluorescent zones under ultraviolet light and by radioactivity in a strip scanner. Compound spotted Enzymatic product... Enzymatic product... o-erythro-neopterin... D-three-Neopterin... L-threo-Neopterin.... I Distance of migration cm 12.2 (radioactivity) 12.2 (fluorescence) phosphocellulose columns at exactly the same rate; D-threoneopterin and L-threo-neopterin are also eluted at the same rate, but at a rate slightly slower than that of the erythro isomers. An experiment was performed in which radioactive material from the peak shown in Fig. 6B was mixed with n-three-neopterin and subjected to rechromatography on phosphocellulose. The resulting radioactive peak obtained from the column coincided exactly

8 3996 Biosynthesis of Folic Acid. VII Vol. 242, No. 18 with the peak due to the carrier n-threo-neopterin. Thus, it appears that GTP is converted by the enzyme preparation into at least three pteridine compounds: hydroxymethylpteridine, either D- or L-erythro-neopterin, and either D- or r.-threo-neopterin. The three solvent systems used in Table VI cannot distinguish between erythro and threo isomers of neopterin; however, another solvent system (isopropyl alcohol-5% boric acid), which will separate these isomers was used (18), although neither this solvent nor any others that have been tried will separate the D and L forms of either the erythro or threo isomers of neopterin. The tentative identification of a threo isomer of neopterin, as a product formed from GTP, by its behavior on phosphocellulose was confirmed by paper chromatographic analysis summarized in Table VII. DISCUSSION The identification of hydroxymethylpteridine and neopterin as products formed enzymatically from GTP provides strong support for the operation of the biosynthetic pathway proposed by Reynolds and Brown (8) for the conversion of guanine nucleotides to dihydropteroate. The conclusion that the dihydro forms of neopterin and hydroxymethylpteridine are the products formed enzymatically from GTP is supported by the following observations: (a) the corresponding oxidized compounds must first be reduced chemically before they can be used enzymatically; and (b) in the conversion of GTP to dihydropteroate no reducing power and no coenzyme involved inreductionneed to be supplied. The oxidized forms of these pteridines are obtained as products only because the dihydro compounds are oxidized during the procedures used for their purification and isolation. The fact that GTP, and no other guanine nucleotide, is used as substrate confirms the results of others (1, 9, 1) that GTP is used directly as the precursor. The previous reports (7,8, 14) that guanosine, GMP, and GDP could be utilized were due to the fact that ATP, included in the reaction mixtures, undoubtedly allows the enzymatic production of GTP from these compounds. Reynolds and Brown (8) suggested that a phosphate ester of dihydroneopterin might be the intermediate formed from GTP. The results presented in this paper provide no information about this question, since all incubated reaction mixtures were treated with phosphatase to dephosphorylate any possible phosphate esters before isolation of products was attempted. This was done because the success of separation of pteridine products on phosphocellulose columns depends on the compounds being present with no negative charge. Phosphate esters are not retained on phosphocellulose columns. However, recent evidence from this laboratory (22) has indicated that the intermediate formed from GTP is a triphosphate ester of neopterin. Thus, the three phosphate residues of GTP are retained during the enzymatic conversion of this compound to neopterin. Evidence is presented in this paper that erythro-dihydroneopterin is formed from GTP, but no method is available to decide whether such a product is of the D or L configuration. It seems reasonable, however, to think that the product is of the D configuration, since GTP is a derivative of n-ribose and would thus yield n-erythro-dihydroneopterin by direct formation from GTP. Production of n-erythro-dihydroneopterin from GTP would mean that the configuration around both of the asymmetric carbon atoms of the 3-carbon side chain would have to be inverted, a possibility t hat seems unlikely. Also, the fact that D-erythro- dihydroneopterin is utilized much more effectively than L-erythrodihydroneopterin (13) for production of dihydropteroate is in favor of the D compound as the true intermediate. The significance of the enzymatic production of the three isomer of neopterin remains unclear. Additional experiments from this laboratory, not described in the Results, have shown that production of this threo compound can be catalyzed by unheated extracts of E. coli, but that heated extracts, which retain the ability to catalyze the synthesis of hydroxymethyldihydropteridine from n-erythro-dihydroneopterin, are not effective in catalyzing the formation of the threo compound from the erythro compound. These observations suggest that formation of the threo compound from the erythro-dihydropteridine proceeds via an enzymatic epimerization and, furthermore, that enzymatic conversion of n-erythro-dihydroneopterin to hydroxymethyldihydropteridine does not proceed through the intermediate formation of a threo-dihydroneopterin. It is possible that E. coli needs to make pteridines, for purposes that remain unknown, distinct from the pteridines used for making folic acid, and that the formation of the threo-neopterin represents a step for production of these other pteridines. The fact that a heated enzyme preparation is able to catalyze the formation of hydroxymethyldihydropteridine from D-erythrodihydroneopterin provides strong support for the view that a heat-stable enzyme or enzyme system is responsible for this transformation. This enzyme is undoubtedly identical with the heat-stable component required for the formation of an intermediate in the conversion of n-erythro-dihydroneopterin to dihydropteroate (see Table III). Clearly, from the results presented in this paper, the conversion of n-erythro-dihydroneopterin to hydroxymethyldihydropteridine does not proceed through the intermediate formation of 2-amino-4-hydroxydihydropteridine. The most likely possibility is that this transformation occurs directly with the removal of 2 of the 3 carbons of the side chain of dihydroneopterin as a 2-carbon unit. Theoretical considerations suggest that such a 2-carbon unit could be glycolaldehyde. In an attempt to obtain information to support this possibility, we have included glycolaldehyde in reaction mixtures in which, otherwise, conditions were optimal for the conversion of n-erythro-dihydroneopterin- 1% to dihydropteroate. In this way, we hoped to be able to trap any radioactive glycolaldehyde that might be produced. However, it was found that glycolaldehyde, as well as a number of other aldehydes, inhibits the enzymatic transformation of D- erythro-dihydroneopterin to hydroxymethyldihydropteridine. Aldehydes do not inhibit the conversion of the latter compound to dihydropteroate. Incubation of radioactive n-erythro-dihydroneopterin with enzyme, in the absence of glycolaldehyde, did not yield any detectable amount of aldehyde, but it was later found that any aldehyde that would have been produced would not have been detected because of the rapid reaction of aldehydes with degradation products of the sodium hydrosulfite used to reduce the pteridine substrate. Work is being planned to modify the experimental conditions so that it will be feasible to identify the nonpteridine product of the reaction. It was reported by Reynolds and Brown (8) that inorganic phosphate stimulates, and arsenate inhibits, the formation of dihydropteroate from GTP. We have determined that neither phosphate nor arsenate has any effects on the utilization of either n-erythro-dihydroneopterin or hydroxymethyldihydropteridine for the enzymatic production of dihydropteroate. Burg and

9 Issue of September 25, 1967 T. H. D. Jones and G. M. Brown 3997 Brown3 have found that neither arsenate nor phosphate has any 8. REYNOLDS, J. J., AND BROWN, G. M., J. Biol. Chem., 239, 317 effect on the first enzymatic reaction of the pathway, i.e. the (1964). 9. SHIOTA, T., AND PALUMBO, M. P., J. Biol. Chem., 24, 4449 removal of carbon atom 8 of GTP as formate. Therefore, the (1965). stimulatory effect of phosphate must be on one or more enzy- 1. DALAL, F. R., AND GOTS, J. S., Biochem. Biophys. Res. Commatic reactions after the first one of the pathway and before the mm., 2, 59 (1965). utilization of n-erythro-dihydroneopterin. 11. WEYGAND, F., SIMON, H., DAHMS, G., WALDSCHMIDT, M., SCHLIEP, H. J., AND WACKER, H., Angew. Chem., 73, 42 (1961). Acknowledgment-The authors wish to thank Mrs. Barbara. 12. BRENNER-HOLZACH, O., AND LEUTHARDT, F., Helv. Chim. Schwartz for her skillful technical assistance during these Acta, 44, 148 (1961). investigations. 13. JONES, T. H. D., REYNOLDS, J. J., AND BROWN, G. M., Biochem. Biophys. Res. Commun., 17, 486 (1964). REFERENCES 14. MITSUDA, H., SUZUKI, Y., TADERA, K,. AND KAWAI, F., J. Vitaminot., 12, 192 (1966). 1. BURG, A. W., AND BROWN, G. M., Biochim. Biophys. Acta, 117, 15. LOWRY,. H., ROSEBROUGH, N. J., FARR, A. L., AND RAN- 275 (1966). DALL, R. J., J. Biol. Chem., 193, 265 (1951). 2. BROWN, G. M., WEISMAN, R. A., AND MOLNAR, D. A., J. Biol. 16. WALLER, C. W., GOLDMAN, A. A., ANGIER, R. B., BOOTHE, J. Chem., 236, 2534 (1961). H., HUTCHINGS, B. L., MOWAT, H. J., AND SEMB, J., J. 3. WEISMAN, R. A., AND BROWN, G. M., J. Biol. Chem., 239, 326 Amer. Chem. Sot., 72, 463 (195). (1964). 17. REMBOLD, H., AND METZGER, H., Chem. Ber., 96, 1395 (1963). 4. SHIOTA, T., AND DISRAELY, M. N., Biochim. Biophys. Acta, 18. REMBOLD, H., AND BUSCHMANN, L., Chem. Ber., 96,146 (1963). 62, 467 (1961). 19. REMBOLD, H., AND BUSCHMANN. L., Ann. Chem., Justus 5. BOCCHIERI, S., AND KOFT, B., Bacterial. Proc., 74 (1965). Liebigs, 662, 72 (1963). 6. ORITZ, P. J., AND HOTCHKISS, IZ. D., Biochemistry, 6,67 (1966). 2. FREIDKIN. M.. CRAWFORD. E. J.. AND MISRA., D.., Fed. Proc.. 7. R.EYNOLDS, J. J., AND BROWN, G. M., J. Biol. Chem., 237, 21, 176 il96i). PC2713 (1962). 21. MACLEAN, F. I., FORREST, H. S., AND MYERS, J., Biochem. Biophys. Res. Commun., 18, 623 (1965). 3 A. W. Burg and G. M. Brown, unpublished observations. 22. BURG, A. W., AND BROWN, G. M., Fed. Proc., 26, 343 (1967).