©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Investigation of the Early Steps of Molybdopterin Biosynthesis in Escherichia coli through the Use of in Vivo Labeling Studies (*)

(Received for publication, July 22, 1994; and in revised form, November 14, 1994)

Margot M. Wuebbens K. V. Rajagopalan (§)

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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.


INTRODUCTION

With 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(1, 2, 3) . 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 (4) . In addition, the isomeric state of the dihydro pterin ring of the active cofactor may vary from enzyme to enzyme(5, 6, 7) .


Figure 1: Structures of molybdopterin, precursor Z, and compound Z.



Exploration of the pathway of biosynthesis of the molybdenum cofactor has been facilitated by the existence of pleiotropic mo mutants (8) 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; (8) ) have clarified the final steps of cofactor biosynthesis in this organism. In the terminal step of molybdopterin synthesis, the desulfo molybdopterin intermediate precursor Z (9, 10) is converted to molybdopterin through the action of molybdopterin synthase (previously termed ``converting factor'')(11, 12) . 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(11, 12) . 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)(13) .

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(10) . Precursor Z accumulates in the E. coli mutants moeB and moaE(9, 10, 14) and is also present in the urine of group B cofactor-deficient humans(15) . Precursor Z may be assayed either by the appearance of fluorescent compound Z following oxidation of a sample (9, 14) or by the production of active molybdopterin upon incubation with a source of molybdopterin synthase(11, 12, 15) .

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 (H(4)B) (^1)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(16, 17, 18, 19, 20, 21, 22) . These findings were clarified by the purification and characterization of the enzyme GTP cyclohydrolase I, which converts GTP to the reduced pterin, H(2)NTP(23, 24) . 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(22) . 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 H(2)NTP, which then serves as the common biosynthetic intermediate for both the folates and H(4)B.


Figure 2: Initial steps in the known pathways of pteridine biosynthesis. Top, conversion of GTP to H(2)NTP 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.



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(25) . 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(25) . 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 H(2)NTP 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 METHODS

Chemicals and Reagents

Ammonium chloride, sodium chloride, KH(2)PO(4), Na(2)HPO(4), and glucose were from Mallinckrodt. CaCl(2) and KMnO(4) were from J.T. Baker Chemical Co. Thiamine hydrochloride, QAE-Sephadex, and ribose were from Sigma. MgSO(4), NaIO(4), 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 geq98%.

Dephosporylation of [8,5`-^3H]GDP

After evaporation to dryness in air, the GDP was resuspended in 250 µl of H(2)O followed by the addition of 25 µl of 0.5 M MgCl(2) and 20 µl of alkaline phosphatase (2 mg/ml of H(2)O). After overnight incubation, the alkaline phosphatase was inactivated by heating the solution in a boiling H(2)O bath for 45 s.

Growth of moeB Cells

Labeling 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 NH(4)Cl, 3.0 g of KH(2)PO(4), and 6.0 g of Na(2)HPO(4)/liter) supplemented with 15 ml of 20% glucose, 5 ml of 0.1 M CaCl(2), 1 ml of 1.0 M MgSO(4), 1 ml of 2 mg/ml thiamine, and 1 ml of a 1:10 dilution of Vogel Medium N stock trace element solution(26) . 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 Z

The 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% I(2), 2% KI in H(2)O 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 times 10.0-cm column of QAE-Sephadex (acetate form) in three batches. After washing with 150 ml of H(2)O 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 times 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 H(2)O, 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 times 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 Z

The remainder of the compound Z sample was adjusted to pH 12-13 with 1 N NaOH. An excess of 25 mM KMnO(4) in H(2)O 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 KMnO(4) was reduced by the addition of 100 µl of 95% ethanol, and the precipitated MnO(2) 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 NH(4)OH 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 (10) and spectrally quantitated based on a molar extinction coefficient at 344 nm of 7410(27) . Aliquots were then removed for liquid scintillation counting and determination of specific radioactivity.

Formation of Pterin from pt-6-COOH

The 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 H(2)O 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(27) .

Isolation of Pterin and Bulk pt-6-COOH from Cells and Media Labeled with [8-^14C]Guanosine

Pterin 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 NH(4)OH. 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 KMnO(4), the entire sample was filtered through two layers of Whatman paper on a Buchner funnel to remove solid MnO(2). 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.


RESULTS

Purification of Compound Z

Although 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(9) . 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(9) . Le Van et al. (28) and Bacher et al. (29, 30) have utilized in vivoC 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 Z

In 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 (31, 32) 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 CO(2) to yield free pterin (33) , 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.



Labeling with [U-^14C]Guanosine

Due 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 1shows 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.



To 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]Guanosine

The 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 C-8 carbon of guanosine to an extent comparable to that observed with the uniformly labeled guanosine. In addition, essentially 100% of this label was retained in pt-6-COOH after alkaline permanganate cleavage of the three terminal side chain carbons, while UV cleavage of the final carboxylate carbon resulted in a quantitative loss of all ^14C label.



The possibility that the label observed in these experiments was due to the presence of a small amount of a highly labeled contaminant in the purified compound Z was ruled out for three reasons. 1) Identical results were obtained from compound Z independently purified in three separate experiments. 2) Virtually 100% of the ^14C-specific radioactivity was retained after permanganate cleavage of each compound Z sample and subsequent HPLC purification of the resulting pt-6-COOH. 3) Prior to HPLC purification of the pterin produced by UV cleavage of pt-6-COOH in the third experiment listed in Table 2, an aliquot of the UV-treated mixture was assayed for ^14C. No radioactivity was present in this sample (data not shown), indicating that all of the pt-6-COOH label had been converted to volatile CO(2) as expected from the degradation scheme shown in Fig. 3.

These results suggested that, unlike the synthesis of all other pterins, during molybdopterin biosynthesis the C-8 carbon of the guanosine precursor is retained and transferred directly to the C-1` position of the pterin side chain. However, to establish with certainty that the results observed with [8-^14C]guanosine were not due to an alteration in general pterin biosynthesis specific to the moeB mutant, the labeling patterns of free pterin and the pt-6-COOH derived from all cellular 6-substituted pterins was examined. The results shown in Table 3demonstrate that the pterin was unlabeled and that the pt-6-COOH had a specific radioactivity which was only 8.3% of that observed for the compound Z purified from the same culture (trial 3, Table 2). UV cleavage of this pt-6-COOH also produced a total loss of label. These results indicate that the activities of the enzymes involved in the synthesis of other pterins in E. coli are not altered in the moeB mutant. The small amount of labeled pt-6-COOH was most likely derived from the photolysis of precursor Z in the medium.



Labeling with [8,5`-^3H]Guanosine

The results obtained from growth of moeB on [U-^14C]guanosine indicated that radiolabel from the ribose carbons of this molecule was transferred to the side chain of precursor Z, but did not reveal which of these ribose carbons were retained in which positions of the pterin side chain. Since no other [^14C]guanosine compounds were available, tritiated guanosine was employed to further clarify the mechanism of conversion of a guanosine precursor to molybdopterin. Compound Z purified from media supplemented with 8-^3H guanosine contained no label (data not shown), indicating that unlike the C-8 carbon, the C-8 proton of guanosine is not retained during cofactor biosynthesis. Growth of moeB cells on [8,5`-^3H]guanosine, however, did result in transfer of label to compound Z as seen in Table 4. Since the proton from the C-8 position is not retained, the observed label must have originated from one or both of the 5` methylene protons of the ribose. Further, the almost quantitative loss of radioactivity upon cleavage of the three terminal side chain carbons of compound Z with KMnO(4) indicated the labeled proton(s) must be associated with the C-2`, C-3`, or C-4` of compound Z. Again, while growth on both ribose and glucose resulted in a decrease of compound Z specific radioactivity when compared to growth on glucose alone, the distribution of the radioactivity within the molecule remained unchanged.




DISCUSSION

The results obtained from these studies have provided the first available information about the early steps in the biosynthesis of the molybdopterin portion of the molybdenum cofactor in E. coli, which can be summarized as follows:

1) A guanosine derivative serves as the initial biosynthetic precursor as demonstrated by the transfer of label from [U-^14C]guanosine to precursor Z, the final molybdopterin intermediate. This aspect of molybdopterin biosynthesis is shared with both of the known pteridine biosynthetic pathways.

2) Both the ribose and ring carbons of the guanosine are utilized in this synthesis since cleavage of the side chain carbons resulted in a loss of specific radioactivity from compound Z purified from culture of moeB cells on [U-^14C]guanosine. In this respect, molybdopterin synthesis resembles folate synthesis rather than riboflavin synthesis.

3) The C-8 carbon of the guanosine precursor is retained and incorporated as the first carbon of the molybdopterin side chain. This aspect of molybdopterin synthesis is distinct from either of the other two pathways and indicates that molybdopterin biosynthesis represents a novel route for pteridine synthesis within E. coli. Thus, it is unlikely that early precursors in the previously identified pteridine biosynthetic pathways (i.e. H(2)NTP or ARAPP) can serve as common intermediates for molybdopterin biosynthesis. This conclusion is supported by evidence that indicates that some human cell lines which are deficient in GTP cyclohydrolase I activity, and therefore do not synthesize H(4)B, do actively express the molybdopterin containing enzyme, sulfite oxidase(34) .

Degradative analysis of the U-^14C-labeled compound Z suggested that only one of the three terminal side chain carbons is derived from the guanosine precursor. The average experimental retention of 86.7% of ^14C label shown in Table 1for the cleavage of compound Z to pt-6-COOH was within 1% of that theoretically expected for the loss of a single, labeled carbon from a total of eight labeled carbons (87.5%). In addition, the proportion of label retained after cleavage of the remaining side chain carbon by UV illumination correlated well with the theoretical loss of a single, labeled carbon from fully labeled pt-6-COOH (85.5 versus 85.7% retention of label) and two labeled carbons from the original compound Z (74.1 versus 75.0% retention). Unfortunately, from these experiments it was not possible to ascertain which of the three terminal side chain carbons had been labeled. Since the C-2` carbon of compound Z is contiguous with the remaining labeled carbons, it would at first appear reasonable to assume that cleavage of this carbon atom by permanganate treatment could have accounted for the observed loss of label. However, it is then difficult to imagine how a significant amount of label from [8,5`-^3H]guanosine could also have been incorporated into this position.

An alternative explanation for the labeling data reported in this work is presented in Fig. 4. By this scheme, molybdopterin biosynthesis begins with a phosphorylated guanosine molecule. The initial reactions are hydrolysis of the guanine ring and linearization and rearrangement of the ribose group to yield a formamidopyrimidine 1`-deoxy-2`-ketopentose phosphate intermediate similar to that proposed for the GTP cyclohydrolase I reaction(22, 23, 24) . At this point, the formyl group derived from the C-8 guanine carbon is transferred to the C-2` carbon with concomitant cleavage of the ribose backbone between the C-2` and C-3` positions to form a glyceraldehyde phosphate-type molecule from the three terminal carbons. Reattachment of this moiety to the original C-8 carbon and closure of the pterin ring by elimination of H(2)O yields a phosphorylated dihydropterin with a four-carbon, six-alkyl side chain, which cyclizes to precursor Z, possibly with the loss of one or more phosphates from the side chain. In this pathway, all of the original guanosine carbons are retained in the final product. The loss of only 13.3% of the label upon cleavage of the three terminal side chain carbons of compound Z purified from U-^14C-labeled cultures could then be reinterpreted as the consequence of equilibration between the transient phosphorylated three-carbon side chain intermediate generated during the reaction, and the unlabeled cellular pools of this molecule. Permanganate cleavage of compound Z would then result in the loss of three weakly labeled carbons rather than the loss of a single, highly labeled carbon. In the absence of a suitable alternative route for the cleavage of the side chain carbons of compound Z, however, it is not possible to state with certainty which of the three terminal side chain positions was labeled by [U-^14C]guanosine during molybdopterin biosynthesis.


Figure 4: Possible pathway for the early steps of molybdopterin biosynthesis in E. coli.



In theory, the fate of each individual guanosine carbon during molybdopterin biosynthesis could be determined by characterization of the compound Z purified from cells grown on media supplemented with guanosine labeled only at that specific carbon. This would be particularly useful for investigating the fate of the ribose carbons of the guanosine precursor during molybdopterin synthesis. Unfortunately, the guanosine derivatives utilized for the experiments reported here are the only ^14C- or ^3H-labeled guanosine derivatives which are currently commercially available, hampering further characterization of molybdopterin biosynthesis by the method described here. Nonetheless, the data obtained by this method have yielded the first available information regarding the early steps in the biosynthesis of the molybdopterin cofactors as well as identified a unique pathway for pterin biosynthesis in microorganisms.


FOOTNOTES

*
This work was supported by Grant GM00091 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 919-684-3120; Fax: 919-684-8919.

(^1)
The abbreviations used are: H(4)B, tetrahydrobiopterin; H(2)NTP, 7,8-dihydroneopterin triphosphate; ARAPP, 2,5-diamino-6-ribitylaminopyrimidine 5`-phosphate; pt-6-COOH, pterin-6-carboxylic acid; HPLC, high performance liquid chromatography.


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