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(Received for publication, July 22, 1994; and in revised form, November 14,
1994) From the
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
[ 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
Figure 2:
Initial steps in the known pathways of
pteridine biosynthesis. Top, conversion of GTP to
H
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 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.
HPLC purification employed
successive chromatography on an Alltech 10-µm C-18 column (4.6
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
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.
Figure 3:
Degradation scheme for compound Z
resulting in sequential cleavage of the side chain carbons to produce
free pterin.
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-
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 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-
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- 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- 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 Degradative analysis of the
U- 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
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
Volume 270,
Number 3,
Issue of January 20, 1995 pp. 1082-1087
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
C]guanosine to moeB cultures produced
compound Z labeled in both the ring and the side chain. Growth on
[8-C]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`-
H]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.
B) (
)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
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 HNTP, which
then serves as the common biosynthetic intermediate for both the
folates and HB.
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.
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.
Chemicals and Reagents
Ammonium chloride, sodium
chloride, KHPO, NaHPO,
and glucose were from Mallinckrodt. CaCl and KMnO were from J.T. Baker Chemical Co. Thiamine hydrochloride,
QAE-Sephadex, and ribose were from Sigma. MgSO,
NaIO, 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-C]Guanosine in 10% ethanol with a
specific radioactivity of 500 mCi/mmol was from Research Products
International. [8-C]Guanosine (56 mCi/mmol) and
[8-H]guanosine (15 Ci/mmol) in 2% ethanol were
from Moravek Biochemicals. [8,5`-H]GDP in 50%
ethanol (10 Ci/mmol) was from DuPont NEN. All radiolabeled compounds
had a radioactive purity 
98%.Dephosporylation of
[8,5`-
After evaporation to dryness
in air, the GDP was resuspended in 250 µl of HH]GDPO
followed by the addition of 25 µl of 0.5 M MgCl and 20 µl of alkaline phosphatase (2 mg/ml of
HO). After overnight incubation, the alkaline phosphatase
was inactivated by heating the solution in a boiling HO
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 NHCl, 3.0 g of
KHPO, and 6.0 g of
NaHPO/liter) supplemented with 15 ml of 20%
glucose, 5 ml of 0.1 M CaCl, 1 ml of 1.0 M MgSO, 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-C, 25 µCi of 8-C, 60 µCi of
8-H, or 75 µCi of 8,5`-H) 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% KI in
HO 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 HO 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
HO, 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. 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 in
HO 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 was reduced by the addition of 100 µl of 95% ethanol, and the
precipitated MnO 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 NHOH 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 HO 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-
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 NHC]GuanosineOH. 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., the entire sample was filtered through two layers of
Whatman paper on a Buchner funnel to remove solid MnO. 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.
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.
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 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.
Labeling with
[U-
Due to the poor transport
of phosphorylated molecules into bacterial cells, labeled guanosine
rather than any of the nucleotides was used.
[U-C]GuanosineC]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-C]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 C label upon UV treatment of the pt-6-COOH indicated that
the C-1` carbon of precursor Z had also been labeled by
[U-C]guanosine. These results demonstrate that a
guanine or guanosine derivative is the initial precursor for
molybdopterin biosynthesis.
C]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-
The results of in vivo labeling with [U-C]GuanosineC]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-C]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 C label.
C-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 C. 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 as expected from the degradation scheme shown in Fig. 3. C]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`-
The results obtained
from growth of moeB on [U-H]GuanosineC]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 [C]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-H 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`-H]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 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.
C]guanosine to
precursor Z, the final molybdopterin intermediate. This aspect of
molybdopterin biosynthesis is shared with both of the known pteridine
biosynthetic pathways.C]guanosine. In this respect, molybdopterin
synthesis resembles folate synthesis rather than riboflavin synthesis. 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 HB, do actively
express the molybdopterin containing enzyme, sulfite
oxidase(34) .C-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 C 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`-H]guanosine could also have been
incorporated into this position.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-C-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-C]guanosine during
molybdopterin biosynthesis.
C- or H-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.
)B, tetrahydrobiopterin;
HNTP, 7,8-dihydroneopterin triphosphate; ARAPP,
2,5-diamino-6-ribitylaminopyrimidine 5`-phosphate; pt-6-COOH,
pterin-6-carboxylic acid; HPLC, high performance liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
