(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
[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.
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 (HB) (
)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 H
NTP, which
then serves as the common biosynthetic intermediate for both the
folates and H
B.
Figure 2:
Initial steps in the known pathways of
pteridine biosynthesis. Top, conversion of GTP to
HNTP 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 HNTP 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.
HPLC purification employed
successive chromatography on an Alltech 10-µm C-18 column (4.6
250 mm) equilibrated in 2% methanol, pH 2, 0.5% methanol, pH 2,
0.01 N HCl, and 1 mM ammonium acetate, pH 5. After
concentration to dryness and resuspension in 50 mM ammonium
acetate, pH 5, the absorption spectrum of the compound Z was recorded
and the concentration determined based on a molar extinction of 16,785
at 310 nm. Aliquots were then removed for quantitation of label using a
Beckman LS 1801 scintillation counter. All HPLC analyses were performed
at room temperature and utilized a Hewlett-Packard 1090 solvent
delivery system. Eluting material was monitored for absorbance with a
Hewlett-Packard 1040A diode array detector and for fluorescence with a
Hewlett-Packard 1046A programmable fluorescence detector.
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, 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.
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-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.
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 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.
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-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.
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-C]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-C]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. HNTP 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
B, do actively
express the molybdopterin containing enzyme, sulfite
oxidase(34) .
Degradative analysis of the
U-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.
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 HO 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.
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 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.