THE JOURNAL cm BIOLOGICAL CHEMISTRY

Vol. 236, No. 9, September 1961

Printed in 7J.S.h

The Biosynthesis of Folic Acid

I. SUBSTRATE AND COFACTOR REQUIREMENTS FOR ENZYMATIC SYNTHESIS BY
CELL-FREE EXTRACTS OF ESCHERICHIA COLI*

GENE M. BROWN, ROBERT A. WEIsMAN,t AND DONNA A. MOLNAR

From the Division of Biochemistry, Department of Biology, MassachusettsInstitute

of Technology, Cambridge 39, Massachusetts

(Received for publication, April 6, 1961)

The formation of folic acid compounds from pteridines and the oxidized forms, a finding that led Brown et ~2. (7) to suggest
p-aminobenzoic acid by whole bacterial cells has been studied by that either dihydro- or tetrahydropteroic acid, rather than
Korte et al. (1, 2), who reported that C%xanthopterin (2-amino- pteroate, is the true intermediate in folate biosynthesis. Re-
4,6-dihydroxypteridine) could be incorporated into folic acid cently, Jaenicke and Chan (8) obtained evidence which supports

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by several bacterial species, and by Weygand et al. (3), who this suggestion by showing that synthetic 2-amino-4-hydroxy-6-
found that certain strains of bacteria can be adapted to utilize hydroxymethyldiiydropteridine was utilized effectively for
2.amino-4-hydroxy-6-formylpteridine for folate production. folate synthesis by extracts of E. coli.
The initial experiments concerned with folate synthesis in cell- The present paper describes in detail the folate-synthesizing
free extracts were done by Katunuma.1 His evidence indicated enzyme system of E. coli with regard to effectiveness of various
that extracts of Mycobacterium awium could convert either compounds as substrates, cofactor requirements, nature of
xanthopterin or 2-amino-4-hydroxy-6-carboxypteridine and p- products formed, and possible reaction sequence.
aminobenzoylglutamic acid to folate. Evidence was also
presented to show that these extracts were able to catalyze the EXPERIMENTAL PROCEDURE

formation of p-aminobenzoylglutamic acid from p-aminobenzoic IMaterials-Crystalline ATP, DPN, and TPN were purchased
acid and glutamate with p-aminobenzoic acid-adenylate as an from the Pabst Laboratories; DPNH, FAD, and alcohol de-
intermediate in the system. Somewhat later, Shiota (4) found hydrogenase from Sigma Chemical Company; thiamine pyro-
that extracts of Lactobacillus arabinosus contained enzymes phosphate from Merck and Company, Inc.; p-aminobenzoic acid
which, in the presence of adenosine triphosphate, could utilize as from Fisher Scientific Company; folic acid and sodium ascorbate
substrate either 2-amino-4-hydroxy-6-hydroxymethylpteridine or from the California Corporation for Biochemical Research;
2-amino-4-hydroxy-6-formylpteridine (or the corresponding potassium pyruvate from Mann Research Laboratories; and
tetrahydro forms of these compounds) for the synthesis of either Darco G-60 charcoal from the Atlas Powder Company. Lederle
pteroic acid (from p-aminobenzoic acid) or folic acid (from p- Laboratories Division of American Cyanamid kindly supplied
aminobenzoylglutamic acid). Brown (5) found that extracts of generous amounts of the following compounds: ABG,2 pteroic acid
Escherichia coli contained a similar enzyme system for the (70 y. pure), aminopterin, amethopterin, pteridine, 6-formyl-
synthesis of either pteroate or folate. The E. coli system could pteridine, 6-hydroxymethylpteridine, 6-hydroxypteridine (xan-
utilize as substrate only 2-amino-4-hydroxy-6-formylpteridine or thopterin) , and 6-carboxypteridine. 6-Formylpteridine and 6-
reduced forms of this compound and 2-amino-4-hydroxy-6- hydroxymethylpteridine were also prepared by the procedure of
hydroxymethylpteridine (6, 7) ; reduced diphosphopyridine Waller et al. (9) and used in some of the experiments to be de-
nucleotide and flavin adenine nucleotide, as well as adenosine scribed. The prepared compounds were judged from absorption
triphosphate, also were necessary components of the reaction spectra to be about 50% pure.
mixtures (7). Because p-aminobenzoic acid was a much more Reduction of Pteridines-Folic acid, pteroic acid, 6-formyl-
effective substrate for pteroate synthesis than was p-amino- pteridine, and 6-hydroxymethylpteridine were reduced to the
benzoylglutamic acid for folate synthesis, it was postulated that corresponding tetrahydro compounds by a catalytic hydrogena-
pteroic acid (or a reduced form of pteroate) is an intermediate in tion procedure suggested by Blakley (10). In this procedure,
the biosynthesis of folate in E. coli (6,7). The reduced forms of the pteridine (5 to 10 mg) was dissolved in 5.0 ml of 0.1 N
pteridines appeared to be used more effectively as substrate than NaOH, and reduction was carried out with hydrogen at room
* This work was supported by Research Grants G-4580 from the temperature with vigorous stirring in the presence of PtOz (equal
National Science Foundation and A-3442 from the National Insti- to the weight of pteridine being reduced) as catalyst. Under
tutes of Health of the United States Public Health Service. these conditions, approximately 2 moles of Hz were taken up per
t Sunnorted bv a Coonerative FellowshiD from the National
Science koundati”on (1959-1960) and a pre-doctoral fellowship from 2 The abbreviations used are: pteridine, 2-amino-4-hydroxy-
the National Institutes of Health (1960-1961). pteridine; formyl, hydroxymethyl, hydroxy, carboxy, and methyl,
1 The text of an address given before the 32nd Congress of the when used with nteridine, will refer to the corresponding 6-pteri-
Japanese Biochemical Association, Kyoto, July, 1957. Dr. Katu- dine compound -(e.g. formylpteridine will be used to denote 2-
numa was kind enough to send a copy of this address to the au- amino-4-hydroxy-6-formylpteridine); ABG, p-aminobenzoylglu-
thors. tamic acid.

2534
September 1961 G. M. Brown, R. A. Weisman,
mole of pteridine added. The tetrahydropteridine compounds
were stored in aqueoussolution under hydrogen at -20” until
they were used.
Dihydrofolate and dihydropteroate were prepared by reduc-
tion with dithionite by the method of Futterman (11).
Microbiological Assays-Enzymatically produced folic acid
compounds were determined by microbiological assay with
Streptococcus jaecalis (ATCC No. 8043). Growth wasestimated
turbidimetrically with a Coleman Jr. spectrophotometer(660-
my wave length). In this assay,the generalmethodsof Teply
and Elvehjem (12) were followed except for the following
modifications: (a) sodiumascorbate,pH 7.0 (1.2 mg per ml), was
addedto the double strength basalmedium,and (b) the samples
to be assayedwere diluted to the proper concentrationsfor assay
in sterile ascorbate (6 mg per ml, pH 7.0) and dispensedasep-
tically to assaytubes containing previously autoclaved medium.
A similar procedure wasoriginally usedby Silverman et al. (13)
to prevent destruction of tetrahydrofolate so that it could be
determinedby assaywith Leueonostoc citrovorum. In the present
work, by following the procedure described,reduced forms of
folate and pteroate (which might be formed as products by the
enzyme system) were prevented from beingdestroyed and there-
fore contributed activity in the assay.
Under the assayconditions describedabove, compoundsthat
are active in the “folic acid” assay with S. jaecalis are folate,
dihydrofolate, pteroate, dihydropteroate, tetrahydropteroate,
and various formylated forms of tetrahydrofolate and tetra-
hydropteroate. A comparisonof the activities of someof these
compoundsshowsthat after an l&hour incubation period folate
is the most active of the compoundstested (Fig. 1A). Tetra-
hydropteroate is about half as active as folate, but sincethis
compound is made by catalytic hydrogeneration of pteroate,
probably 50% of it is presentasthe inactive isomer. Therefore,
it might be consideredthat the active isomer and folate are
equally active in the assay.
On testing the effectsof varying the incubation period, it was
found that pteroate, but none of the other compounds,became
more active as the incubation time was lengthened. Maximal
growth with pteroate was attained after a 44-hour incubation
period. At this time, folate and pteroate were approximately
equally active at low concentrations; however, the total growth
achieved with higher concentrationsof folate was significantly
greater than that achievedwith higher concentrationsof pteroate
(Fig. 1B). The data of Fig. 1 show alsothat dihydrofolate and
tetrahydrofolate, were about 50% and 16% as active, respec-
tively, as folate. The reasonsfor the relatively low activities of
these reduced compoundsremain unknown. It seemsunlikely
that the low activities were due to destruction of the compounds
during the assayproceduresinceother experimentsperformedin
this laboratory have shown that leucovorin (which is a stable
compound) and tetrahydrofolate were equally active for L.
citrovorum when tested under the same conditions described
above. Dihydropteroate appearedto be about 40% as active
as folate for S. jaecalis with no evident time effect such as that
observed with pteroate; i.e. dihydropteroate was equally active
at 18 and 44 hours. However, the yield of dihydropteroate
obtained by the method of preparation could not be determined
easily sinceno independentmethod wasavailable for determining
the purity of the compound;therefore, no statementcan be made
about how active pure dihydropteroate might be and, thus, this
compoundwasnot included in the curves shownin Fig. 1.
and D. A. Molnar 2535
I I I I
3o- INCUBATION TIME: I6 HOURS-
TETRAHYDRO-
100
I I I I I I I I I I
40 60 00 0 20 40 60 00
0 20
GROWTH FACTOR, MICROMICROMOLES PER IO ML
FIG. 1. Relative activities of folate and related compoundsin
promoting growth of S. faecalis.
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Since folic acid is readily available and is stable (it can be
autoclaved with the mediumwithout lossof activity) it wasused
routinely as the standard in the microbiological assays. All
values to be reported, therefore, are expressedas folate equiva-
lents, althoughin all casesthe productsof the enzymatic reactions
were not folate, but rather consistedof either dihydropteroate,
dihydrofolate, tetrahydrofolate, unknown derivatives (probably
formylated derivatives) of tetrahydrofolate and tetrahydro-
pteroate, or mixtures of the compoundslisted. Thus, the results
to be reported as “folate equivalents” are subject to correction
factors which dependon the nature of the product or products of
the enzymatic reactions and on the activities of such products
relative to the activity of folate.
Paper Chromatographic and Bioautographic Techniques-Paper
chromatograms(Whatman No. 1 paper) were developedby the
ascendingtechnique. Prior to use, the paper was soakedin a
solution containing 6 mg per ml of sodiumascorbate (pH 7.0)
and then dried. The developing solvent in all of the experi-
ments to be describedconsistedof 0.1 M phosphate (K salts)
buffer, pH 7.0, which also contained 6 mg per ml of sodium
ascorbate. The ascorbateon the paperand in the solvent acted
as an antioxidant to prevent the destruction of dihydro and
tetrahydro forms of pteroate and folate.
Zonesof migration were located by bioautography (14). For
this purpose,the developedchromatogramwasplacedin contact
with the surfaceof solid medium (the S. jaecalisbasalmedium
described above solidified with 2% agar and seededwith a
washedculture of S. juecalis) contained as a 5-mm layer in a
sterile 22- x 30-cmPyrex baking dish. After about 5 minutes,
the chromatogramwas removed and the plate wascovered and
incubated for 16 hours at 30”. The resulting growth zones
correspondedto zonesof migration on the original chromatogram
of folate or compoundsthat can replacefolate as a growth factor
for S. jaecalis. The inoculum usedto seedthe plates consisted
of washedcellsfrom a 16- to 20-hour culture grown on a yeast
extract medium. One milliliter of this suspensionwas used to
inoculate 200 ml of the sterile basal mediumbefore preparation
of the plate. The samegeneralprocedurewasfollowed when L.
citrovorum wasusedas the test organism,except that the growth
2536 Biosynthesis of Folic Acid. I Vol. 236, No. 9

TABLE I were placed in water at 37” contained as a 4- to 6-cm layer in the

Enzymatic formation of ‘ifolate” by cell-free extracts of E. coli
The complete reaction mixture contained: ABG, 0.067 mM;
ATP, 3.3 mM; DPNH, 0.067 mM; MgC12,6.7 mM; mercaptoethanol,
67 mM; and 0.2 ml (10 mg of protein) of crude extract of E. coli,
in a total volume of 1.5 ml of 0.07 M phosphate (K salts) buffer,
pH 7.0. Pteridines were added to reaction mixtures as shown at
concentrations of 0.067 mM. Incubation was for 3 hours at 37”
under anaerobic conditions. After incubation, 1 mmole of mer-
captoethanol was added to each reaction mixture to stop the re-
action and also to help preserve reduced products which might be
formed. “Folate” produced was determined by microbiological
assay with S. faecalis.
Reaction mixture
Complete ................................
Complete (stopped at 0 time) ............
Omit ABG ...............................
Omit ATP. ..............................
Omit DPNH .............................
bottom of a desiccator. The desiccator was then evacuated and
filled with hydrogen (nitrogen and helium work almost as well)
and incubated in an air incubator at 37”. In some cases when
solutions either had to be added to or withdrawn from a reaction
mixture under anaerobic conditions, the reaction mixture was
prepared in a centrifuge tube containing a side arm equipped with
a stopcock. A NO-AIR rubber stopper (a product of Champaine
Industries, Inc., St. Louis, Missouri) was used to close off the
opening at the top of the tube, and the tube was then evacuated
and filled with hydrogen. Solutions could then be added or
withdrawn by means of a syringe equipped with a hypodermic
needle; the needle could be pushed through the rubber stopper
and then withdrawn without disturbing anaerobic conditions.
RESULTS
a
3.32 Synthesis of Folic Acid Compounds by Crude Extracts of E. coli-
0 The ability of crude cell-free extracts of E. coli to catalyze the
0 formation of compounds with folate activity for S. fuecalis in the
3.86 presence of ABG is shown in Table I. No compound other than
2.55
ABG needed to be supplied as substrate or cofactor for synthesis

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Complete + formylpteridine. ............ 2.80
Complete + hydroxymethylpteridine. .... 0.52 to occur although DPNH appeared to be somewhat stimulatory.
Complete + carboxypteridine. ........... 0.85 Some of the pteridines that were tested as substrates not only
Complete + hydroxypteridine ............ 2.89 were not required in the system, but also appeared to inhibit
Complete + methylpteridine. ............ 3.40 synthesis significantly (see Table I).
Complete + pteridine .................... 1.36 Other experiments, not shown here, have indicated that for
maximal synthesis of folate equivalents, reaction mixtures should
contain mercaptoethanol (or other antioxidant such as ascorbate)
medium for L. citrovorum (15) was used and incubation of the and incubation should be carried out under anaerobic conditions.
plates was at 37” instead of 30”. Preparation and Enzymatic Activity of Charcoal-treated Ex-
Growth of E. coli and Preparation of Cell-free Extracts-A strain tracts-It was considered likely that the failure to demonstrate a
of E. coli B (kindly supplied by Dr. S. E. Luria) which is resistant requirement for a pteridine as substrate and for ATP and pos-
to infection with the bacteriophage T, was used as a source of sibly other cofactors was due to the presence of these compounds
enzymes for the work to be reported. This T1-resistant strain in the crude extracts. The results of efforts made to remove
was used because of some trouble encountered by infection with these compounds from the enzyme preparation led to the adop-
T1 (present as a persistent contaminant in the laboratory) tion of the following procedure for this purpose. Enough of a
during the growth of sensitive strains. Cells were grown at 37” neutralized solution of p-aminobenzoic acid was added to the
(with aeration) in 15 liter amounts contained in 20-liter carboys. crude extract (which contained about 40 mg per ml of protein)
The growth medium contained per liter: Na&IPO+ 10.5 g; to give a solution that was 1 mM with respect to p-aminobenzoic
KH,POI, 4.5 g; NH&I, 1 g; MgSOd * 7Hz0, 0.6 g; Casamino acid. A saturated solution of ammonium sulfate (adjusted to
acids (Difco Laboratories), 15 g; glycerol, 10 ml; and CaC12, 30 pH 7.0 with NHIOH) was then added (slowly, with stirring at
mg. Inoculum for each carboy was a l-liter culture grown at 37” 4”) to the extract to give a 90 y0 saturated solution. The protein
with shaking for 16 hours on the medium described above. precipitate was collected by centrifugation, dissolved in a
Growth of the 15-liter cultures was allowed to proceed for about minimal amount of 0.05 M phosphate (K salts) buffer (pH 7.0)
4 hours; the cells were harvested (Stock centrifuge; Wilhelm which also contained p-aminobenzoic acid (1 mM), and the solu-
Stock Maschinenbau, Marburg, Western Germany), washed tion was dialyzed overnight at 4” against at least 200 volumes of
once with 0.9% NaCl solution, recentrifuged, and the moist cell the phosphate-p-aminobenzoic acid buffer. After dialysis,
cake was frozen by immediate immerson in liquid nitrogen. The more p-aminobenzoic acid was added to give a final concentration
frozen cells were then ruptured with the use of a Hughes press of 2 mM, and 200 mg of Darco G-60 were then added per 10 ml of
(Shandon Scientific Company, Ltd., London); the broken cells enzyme preparation (containing about 30 mg per ml of protein).
were suspended in 2 to 3 volumes of 0.05 M Tris buffer (pH 8.0), The suspension was stirred for 10 minutes at 4” and centrifuged
treated with DNase (to reduce the viscosity of the suspension), to remove the charcoal. The clear supernatant solution will be
and centrifuged at 105,000 X 9 for 1 hour. The clear superna- referred to as the “charcoal-treated extract” in the remainder of
tant extract was saved an used as a source of enzymes. of this paper. In the procedure described above, the p-amino-
Protein-Protein was estimated by the method of Lowry et al. benzoic acid was added to the extract as a protective agent. In
(16) with the use of bovine serum albumin (Pentex, Inc.) as the the absence of p-aminobenzoic acid, little or no enzymatic
standard. activity remained after the charcoal treatment. Other experi-
Anaerobic Incubations-In all of the experiments to be de- ments have revealed that ,4BG can serve as a protective agent al-
scribed, incubations of enzyme preparations with substrates were most as well as p-aminobenzoic acid, however, p-aminobenzoic
carried out under anaerobic conditions. For this purpose, reac- acid was used routinely for this purpose because it is more read-
tion mixtures (prepared in 12- to 15-ml centrifuge or test tubes) ily available in large quantities.
September 1961 G. M. Brown, R. A. Weisman, and D. A. Molnar 2537

The substrate and cofactor requirements for the synthesis of TABLE II

“folate” by a charcoal-treated extract are shown in Table II. Substrate and cofactor requirements for “jolate” synthesis
ATP, DPNH, FAD, Mg++, and a source of pteridine (added as with charcoal-treated extracts
formylpteridine) were all required for maximal production of The complete reaction mixture contained: formylpteridine,
folate. No source of ABC needed to be added, as the enzyme 0.067 mM; ATP, 3.3 mM; DPNH, 0.067 miu; FAD, 0.056 mM; MgC12,
preparation contained large amounts of p-aminobenzoic acid and 6.7 mM; mercaptoethanol, 67 mM; and 0.2 ml (about 6 mg of pro-
results to be presented later show that p-aminobenzoic acid can tein) of charcoal-treated extract (containing about O-4 pmole of
p-aminobenzoic acid) in a total volume of 1.5 ml of 0.07 M phos-
be used in place of ABG. Some variations have been noted in the
phate (K salts) buffer, pH 7.0. Incubation and post-incubation
ATP and FAD requirements depending on which of several
treatment were as described in Table I.
separate preparations of charcoal-treated extracts was used as a
source of enzymes. With some preparations, ATP, FAD, or Reaction mixture Folatee~~~dalents
both, were absolute requirements,. and with other preparations
these compounds were only stimulatory. Absolute require- M
ments for a pteridine compound, pyridine nucleotide, and Mg++ Complete..................................... 2.02
have been observed with all of the preparations of charcoal- Omit FAD.. . 0
treated extracts. Omit DPNH. 0
Comparison of p-Aminobenzoic Acid and ABG as Substrates- Omit ATP.................................... 1.23
Shiota (4) reported that a folate-synthesizing enzyme system Omit formylpteridine.. 0
from L. arabinosus could utilize as substrates either p-amino- Omit MgClz.. . / 0
benzoic acid (to yield pteroic acid) or ABG (to yield folic acid).
A comparison of the activities of these two compounds as sub-

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P-AEG. rnp MOLES
strate in the E. coli system (Fig. 2) indicated that p-amino- i!2 0 50 100 150 20 300 400 500
benzoic acid was utilized much more effectively than ABG. I I I r
2 7.0-
TPN, thiamine pyrophosphate, and pyruvate were included in
the reaction mixtures as a result of the finding (discussed in a ::
later section of this paper) that these compounds stimulated s 6.0-
ii 0
“folate” production. The enzyme preparation used in these
experiments was a charcoal-treated extract from which the p-
aminobenzoic acid had been removed by dialysis for a total of 24
hours against 6 changes of 0.05 M phosphate buffer, pH 7.0. 8
Fig. 2 also shows that at very high concentrations p-aminobenzoic 0
acid becomes inhibitory. The products of enzymatic action in
these experiments were found by bioautographic techniques (see
Fig. 3) to be dihydropteroic acid (from p-aminobenzoic acid)
and dihydrofolic acid (from ABG). In the solvent system used,
both pteroate and dihydropteroate moved only slightly; however,
one can distinguish between these two compounds with tube
assays because the activity of pteroate increases with increased ii1
100 200 300
incubation times (see Fig. 1) whereas that of dihydropteroate
P-AB, i-n/~ MOLES
shows no such time effects. The activity of the enzymatic
product was not affected by the length of incubation, a fact that, FIG. 2. A comparison of the effectiveness with which p-amino-
benzoic acid (p-AB) and ABG (p-ABG) are used for enzymatic
when considered along with the chromatographic behavior of the synthesis of “folate.” Reaction mixtures and incubation condi-
compound, indicates that the product was dihydropteroate. tions were as described in Table III except that a dialyzed char-
These results suggest that a dihydropteridine compound is the coal-treated extract was used as a source of enzymes and p-amino-
substrate that reacts with p-aminobenzoic acid to yield di- benzoic acid or ABG was added to reaction mixtures as indicated.
hydropteroate (or with ABG to yield dihydrofolate).
Pteridine Compounds Active as Substrates-Of several pteridines tetrahydropteridine used in comparisons given in Fig. 4 was also
(which were substituted on the 6-position with various groups) produced by catalytic hydrogenation, this compound should also
which were tested, only formyl- and hydroxymethylpteridine consist of two isomers, only one of which might be expected to be
were active as substrates for the formation of folic acid com- active. If this is true and if the indicated corrections are then
pounds; formylpteridine was many times more active than made in comparing activities of pteridine compounds, the active
hydroxymethylpteridine (see Table III). Also, hydroxymethyl- isomer of hydroxymethyltetrahydropteridine would be more than
tetrahydropteridine was several times more active as substrate twice as active as either formylpteridine or the active isomer of
than the nonreduced compound (Table III). A comparison of formyltetrahydropteridine. These considerations are consistent
the relative activities of pteridine compounds, given in Fig. 4, with the idea that reduced forms of pteridine compounds are
shows that formylpteridine was about twice as active as formyl- more direct precursors of folate than are the fully oxidized forms.
tetrahydropteridine, probably because the latter compound was Support for this tentative conclusion was provided by experi-
prepared by reduction by catalytic hydrogenation, a procedure ments which showed that in the presence of either formyltetra-
which would be expected to produce two isomers (caused by hydropteridine or hydroxymethyltetrahydropteridine “folate”
configuration around carbon 6 of the pteridine ring), only one of synthesis increased linearly with time and that the straight line
which would probably be active. Since the hydroxymethyl- which could be drawn passed through the origin (Fig. 5) (i.e.
2538 Biosynthesis of Folk Acid. I Vol. 236, Eo. 9

which was formed, it was thought that by adding a DPNH-

0.9 generating system it might be possibleto reduce the DPNH
requirement to a stoichiometric or catalytic level. However,
0.8
when the DPNH concentration was reducedto 20 mpmolesper
0.7 reaction mixture, no “folate” synthesistook place (with formyl-
1
pteridine as substrate) even in the presenceof alcohol dehy-
0.6
t- -1 drogenaseand ethanol asa DPNH-generating system. Further
investigationsrevealedthat synthesiscouldbe restoredby adding
TPN to the reaction mixture and also that the addition of
thiamine pyrophosphateand pyruvate increasedthe amount of
“folate” formed. These results are summarizedin Table IV.
Under the conditions employed in these experiments, both
DPNH and TPN wererequired for maximal synthesisand DPN
could be substitutedfor DPNH with no decreasein formation of
product as long as thiamine pyrophosphateand pyruvate were
present in the reaction mixture; in the absenceof these two
FIG. 3. A drawing of a bioautogram which shows the nature of compounds,DPN was less active than DPNH (Table IV).
products formed from p-aminobenzoic acid and ABG by charcoal- Theseresultssuggestthat the TPN requirement wasmet in the
treated extracts. Reaction mixtures were the same as those presenceof the higherconcentrationsof DPNH or DPN probably
described in Fig. 2. ReactionMixture 1 contained p-aminobenzoic
acid (0.013 mM) as substrate and Reaction Mixture d contained by the enzymatic formation of TPN from DPN and ATP.

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ABG (0.067 mM). After incubation, about 0.005 ml of each reac- Furthermore, these data also suggest that the stimulatory
tion mixture was spotted on paper along with the control com- propertiesof thiamine pyrophosphateand pyruvate werea result
pounds (an amount of each equivalent in activity to about 20 of thesecompoundssupplying an efficient enzymatic system (by
mpg of folate was spotted). The chromatogram was developed
and bioautogramswere preparedwith the use of S. faecalis ac- the oxidation of pyruvate) for the formation of DPNH from
cording to directions given in “Experimental Procedure.” DPN. It was alsofound that the addition of glucose-6-Pand
glucose-6-Pdehydrogenase(a system which efficiently converts
TABLE III TPN to TPNH) to reaction mixtures inhibited “folate” produc-
Efectiveness of various compounds as substrates
pteridine tion, a fact which argues against a requirement for TPNH in the
for “folate” synth.esis system and therefore suggeststhat TPN is required in someas
Reaction mixtures contained: ATP, 3.3 mM; DPNH, 0.067 mM; yet unexplainedway. Although DPNH and TPN are active in
TPN, 0.067 mM; FAD, 0.056 mar; MgC12,6.7 mr.r; mercaptoethanol, the lower concentrations (20 mpmoles of each per reaction
67 mM; thiamine-PP, 0.013 mM; potassium pyruvate, 1.33 mM; mixture) ‘in the presence of thiamine pyrophosphate and pyruvate,
pteridine compound as shown, 0.067 mr,r; and 0.2 ml (about 6 mg in order to insure that pyridine nucleotideswere never limiting,
of protein) of charcoal-treated extract (containing about 0.4 it was decidedto add thesecompoundsto reaction mixtures at
pmole of p-aminobenzoicacid) in a total volume of 1.5 ml of 0.07 the higher concentrations(166mCcmoles per reaction mixture) in
M phosphate buffer, PH 7.0. Incubation and post-incubation all of the work not directly concernedwith establishingactivities
treatment were as described in Table I. of DPNH and TPN.
Pteridine added Folatef;rq;$alents

Pg
None ........................................ 0
Formylpteridine .............................. 6.72
Hydroxymethylpteridine ..................... 0.48
Formyltetrahydropteridine. .................. 3.42
Hydroxymethyltetrahydropteridine. .......... 5.52

“folate” synthesis began immediately after incubation had

begun); on the other hand, when formylpteridine was usedas
substrate a lag period of about 40 minutes resulted before
“folate” synthesisbegan (Fig. 5). These results suggestthat
the formylpteridine had to be transformed into perhaps a 0 2.0
reducedpteridine compoundbefore it could be utilized for folate 34
synthesisand that the formation of this new compoundwas the v 1.0
rate-limiting step in the enzyme system when formylpteridine 7
E
wasusedas substrate. 0 20 40 60 80 100 120 140 160 160 200
Nature of the Pyridine NucleotideRequirement-In the initial
PTERIDINE COMPOUND, rnp MOLES
experimentswith the useof charcoal-treatedextracts asa source
of enzymes,DPNH in an amount of 160 mpmolesper reaction FIG. 4. A comparisonof the activities of certain pteridine com-
poundsas substratesfor “folate” synthesis. Reaction mixtures
mixture was added as a source of pyridine nucleotide. Since and incubation conditions were as described in Table III except
this amount is at least 5 times morethan the amount of product that pteridine compounds were added in the amounts shown.
September 1961 G. M. Brown, R. A. We&man, and D. A. Molnar 2539

It was thought that a correlation between cofactor require- TABLE IV

ments and the type of pteridine compound that could be used as Pyridine nucleotide, thiamine pyrophosphate, and pyruvate
substrate might be helpful in establishing cofactor requirements for enzymatic synthesis of “folate”
requirements
for each enzymatic step. Accordingly, formylpteridine, formyl- Reaction mixtures and incubation conditions were as described
tetrahydropteridine, and hydroxymethyltetrahydropteridine in Table III except that only formylpteridine was added as pteri-
were all tested in the presence and absence of individual cofactors. dine substrate to each reaction mixture and pyridine nucleotides
The results of these experiments (summarized in Table V) show were added in the amounts shown.
that for maximal synthesis a full complement of cofactors (i.e. Folate equivalents formed
ATP, FAD, Mg++, DPNH, TPN, and thiamine pyrophosphate) Amount of
Pyridine nucleotide added pyridine
and pyruvate were required only when formylpteridine was nucleotide Absence of Presence of
added thiamine-PP thiamine-PP
added as substrate. The use as substrate of either formyl- or and pyruvate and pyruvate
hydroxymethyltetrahydropteridine. required the presence only
of ATP, Mg++, and TPN, although some synthesis occurred
None................ 0 0 0
with hydroxymethyltetrahydropteridine even in the absence of
DPNH. 100 3.03 4.21
TPN. DPN, when added at relatively high concentrations, was
DPN . 100 2.92 3.68
able to be utilized in place of TPN, probably because it was TPN 100 1.58
converted to TPN. The amount of TPN needed for maximal DPNH+TPN...... 100 of each 3.22 6.44
synthesis from hydroxymethyltetrahydropteridine and the re- DPN + TPN. 100 of each 3.24 6.49
quirement for ATP are shown in Table VI. These results DPNH. 20 1.09 1.21
(Tables V and VI) indicate (a) that FAD, DPNH, and TPN DPN 20 0 1.38

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were required for the enzymatic reduction of the formylpteridine TPN 20 0 0
to another pteridine (probably hydroxymethyldihydropteridine) , DPNH+TPN...... 20 of each 3.03 5.36
(5) that TPN was required probably for the oxidation of tetra- DPN + TPN. .. 20 of each 1.47 5.57
-
hydropteridine compounds to the corresponding dihydro com-
pounds, and (c) that the hydroxymethyltetrahydropteridine TABLE V
preparation was either contaminated with the corresponding Relation of coenzyme requirements to pteridine compound used as
dihydro compound or, prior to utilization, the tetrahydro substrate for enzymatic synthesis of “folate”
compound was partially oxidized nonenzymatically to the Reaction mixtures and incubation conditions were as described
in Table III.
*
5
2
” 0.0
I:Folate equivalents formed with pteridine
added as substrate
0
Coenzyme added to reaction mixture
z Hydrox - Formyl-
f 7.0 methy Y- tetrahydro- Formyl-
FORMYL tetrahydro- pteridine pteridine
TETRAHYDROPTERIDINE
pteridine

/a
I- (rg Pg

HYDROXYMETHYL
0 None.........................
DPNH. . .
3.08
4.04
0.83
1.25
0
0
TETRAHYDROPTERIDINE DPN......................... 3.70 1.13 0
TPN . . 7.12 2.40 0
DPNH + TPN. 7.24 2.16 0
DPNH + TPN + FAD. 7.24 2.16 2.61
DPNH + TPN + FAD + thi.
amine-PP + pyruvate.. .. 7.26 2.38 5.27

FORMYL PTERIDINE
dihydro compound, since some synthesis occurred in the absence
of TPN.
v- Conversion of Reduced Pteroate to Reduced Folate-The results
2?1 470 60 80 100 120 140 160 I80
TIME IN MINUTES
which have been presented suggest that in E. coli dihydropteroate
FIG. 5. The rate of “folate” synthesis as a function of the pteri- is formed relatively efficiently from p-aminobenozic acid and
dine compound added as substrate. The composition of the reac- what is thought to be hydroxymethyldihydropteridine and that,
tion mixtures was as described in Table III. Reaction mixtures therefore, dihydropteroate is an intermediate in the biosynthesis of
were prepared in the special tubes described in “Experimental folate or reduced forms of folate. If this is true, then it might
Procedure.” At zero time, the reactions were started by adding be possible to demonstrate that crude extracts catalyze a reac-
the enzyme preparation (which had been warmed to 37’) to the
otherwise complete reaction mixtures by means of syringe tion whereby dihydropteroate and glutamate are converted
equipped with a hypodermic needle. The reaction mixtures were to a folate compound. Experiments performed with undialyzed
made anaerobic and were warmed to 37” prior to the addition of crude extracts of E. coli showed that both dihydro- and tetra-
the enzyme preparation. Aliquots (0.1 ml) were withdrawn at the hydropteroate were converted to a compound that seemed to be a
times indicated with a hypodermic needle and immediately diluted
derivative of tetrahydrofolate (as observed on a bioautogram
to the proper volumes (in sterile ascorbate at 6 mg per ml) and
added to previously prepared microbiological assay tubes. “Fo- with L. citrouorum as the test organism). No glutamate require-
late” activity was estimated by assay with S. faecalis. ment for synthesis of this compound could be demonstrated.
 Biosynthesis of Folic Acid. I Vol. 236, No. 9

TABLE VI tion of this compound. The new compound was not folate,
Amount of TPN required to stimulate maximal “folate” synthesis tetrahydrofolate, or dihydrofolate (compare Rp values of Fig. 6
Reaction mixtures contained: ATP, 3.3 mru; hydroxymethyl- with those in Fig. 3)) but almost surely was a derivative (perhaps
tetrahydropteridine, 0.067 mM; MgC12, 6.7 mm; mercaptoethanol, containing a l-carbon unit) of tetrahydrofolate since, as far as is
67 mru; 0.2 ml (about 6 mg) of charcoal-treated extract (contain- known, the only folic acid compounds which are active for L.
ing about 0.4 pmole of p-aminobenzoic acid); and TPN as shown citrovorum are those that contain the tetrahydropteridine ring
in a total volume of 1.5 ml of 0.07 M phosphate buffer, pH 7.0. structure. Since the enzyme preparation was relatively crude,
Incubation and post-incubation treatment were as described in
the chances are good that the enzymes and l-carbon donor com-
Table I.
pounds necessary for the formation of tetrahydrofolate deriva-
TPN added to reaction mixture Folate equivalents formed tives were present.
It can also be noted from Fig. 6 that another compound (RF of
)mwle La about 0.50) was formed. Since the formation of this compound
0 2.36 was not glutamate-dependent and since the compound was active
0.02 4.95 for S. faecalis but not for L. citrovorum, it seems likely that this
0.05 5.32
0.10 5.81 compound was an unidentified derivative of either dihydro- or
0.20 5.87 tetrahydropteroate.
0.20 (omit ATP) 2.95 Two possible pathways for the enzymatic formation of the
tetrahydrofolate compound merited consideration: (a) the reac-
tion of tetrahydropteroate with glutamate to yield tetrahydro-
folate directly, and (a) the formation of dihydrofolate from

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diiydropteroate and glutamate followed by reduction to yield
B tetrahydrofolate. To help decide which of these pathways
0.8 operates in the E. coli system, reaction mixtures were prepared

0.6 0.9 A 0
0.6 t

0.7 t

0
0.6 - \ An\
\
\
: )(

0.2 - ,-\
REACTION REACTION TETRA ,- I 1
MIXTURE I MIXTURE 2 HYDRO 0.1 - ‘-’ @ @:::lo
FOLAT E
Q
FIG. 6. A drawing of a bioautogram which illustrates the REACTION REACTION REA TMN FOLATE MHYW‘bbMINO- AMYOPTERIN AHETHOPTI
glutamate-dependent enzymatic conversion of reduced pteroic MIXNREA MIXTURE B MIXTVREC FIXATE PTER,N
acid to a folate compound. Reaction mixtures were prepared to
contain : tetrahydropteroate, 0.024 mM; glutamate (when added), FIG. 7. A drawing of a bioautogram which indicates that di-
0.067 mnil; ATP, 1.3 mM; DPN, 0.067 rnbr; MgCL, 6.7 mM; mercapto- hydrofolate is the initial product formed from dihydropteroate
ethanol, 67 mM; and 0.2 ml (about 10 mg of protein) of crude and glutamate. Reaction mixtures were prepared to contain:
extract of E. coli (dialyzed exhaustively as described in the text), dihydropteroate, about 0.02 mr.r; glutamate (when added), 0.067
in a total volume of 1.5 ml of 0.07 M phosphate (K salts) buffer, mM; ATP, 1.3 mM; DPN, 0.067 mM; MgCL, 6.7 m&r; mercapto-
pH 7.0. Glutamate was added to Reaction Mixture 1 but not to ethanol, 67 mr.r; 0.2 ml of crude extract of E. coli (exhaustively
Reaction Mixture R. Incubation was for 3 hours at 37’ under dialyzed); and either aminopterin or amethopterin, 0.067 mM, in
anaerobic conditions. After incubation, 1 mmole of mercapto- a total volume of 1.5 ml of 0.07 M phosphate buffer, pH 7.0. Post-
ethanol was added to each reaction mixture and 0.005 ml aliquots incubation treatment was as described in Fig. 6.
were spotted, along with control compounds as shown (about 20 Reaction Mixture A contained glutamate and aminopterin;
wg of each), on paper sheets. The chromatograms were de- Reaction Mixture B contained glutamate and amethopterin; Reac-
veloped and bioautograms were prepared with S. faecalis (A) and tion Mixture C contained amethopterin but no glutamate. The
with L. citrovorum (B) as the test organisms. bioautogram (A) was prepared with S. faecalis as the test organ-
ism. The zones indicated with broken lines are inhibition zones
which were visible against a background of faint growth on the
However, after extensive dialysis of the crude extract for 24 hours bioautogram. By contrast the zones of migration of folate com-
against 6 (4 hours each) changes (about 200 volumes each) of pounds appeared as heavy growth zones (diagonal hatching). Note
0.05 M phosphate buffer (pH 7.0), the formation of the new that aminopterin was contaminated with folate. The zones of
compound was shown to be dependent on the addition of gluta- migration of the inhibitors were determined by preparing a bio-
mate to the reaction mixture (Fig. 6). The new compound autogram (B) from a chromatogram on which had been spotted
each of the inhibitors. This bioautogram was prepared with
(Rp of 0.70, Fig. 6) possessed growth promoting activity for S. growth medium to which folate had been added (10 mpg per ml)
faecalis and for L. citrovorum. Both dihydro- and tetrahydro- and with S. faecalis as the test organism. The zones shown in B
pteroate, but not pteroate, could be used as substrate for forma- thus refer to zones of inhibition of growth of S. faecalis.
September 1961 G. M. Brown, R. A. Weisman, and D. A. Molnar 2541

which contained either aminopterin or amethopterin, both of

which are known to inhibit effectively the enzymatic reduction of
dihydrofolate to tetrahydrofolate (11, 17-19). If tetrahydro-
folate is formed directly (Pathway a, above) then this compound,
or a derivative, should be present in the incubated reaction
mistures, whereas if dihydrofolate is the product (Pathway b,
above) then dihydrofolate, but no tetrahydrofolate compound,
should be detected in an incubated reaction mixture. The results
given in Fig. 7 show that under these conditions only dihydro-
folate \vas found; thus, it must be concluded that dihydropteroate
Mg* ATP
had reacted directly with glutamate to yield dihydrofolate. I
Other esperiments have revealed that if tetrahydropteroate is
used as substrate in the presence of the inhibitors, again only
dihydrofolate could be detected as product, a fact which indicates
that tetrahydropteroate was probably converted to dihydro-
pteroate prior to reaction with glutamate. In these experiments,
two types of reaction mistures were prepared, one (containing ;pH2-NHo COOH
aminopterin) served to show that no tetrahydrofolate or deriva-
tives were formed and the other (containing amethopterin) was
prepared to show that dihydrofolate was formed. It was i)H
GLUTAMIC ACID

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necessary to use both inhibitors in this way since, in the solvent l-
system used, aminopterin and dihydrofolate migrated to the
same area on the chromatogram (see Fig. 7) and, thus, any
dihydrofolate that might be formed in the presence of amino-
pterin would not be detected on bioautograms because of the
growth inhibitory properties of aminopterin. The results shown
in Fig. 7 also clearly establish that in the absence of glutamate
and in the presence of amethopterin no dihydrofolate was
produced (Reaction Mixture C).
The enzyme system responsible for the formation of dihydro-
folate from dihydropteroate and glutamate is very labile, and all dOOH
fractionation attempts or treatments with either charcoal or FIG. 8. Proposed pathway for biosynthesis of folate com-
Dowes 1 to remove possible cofactors so far have resulted in the pounds.
complete irreversible inactivation of the system. Until some
method can be devised to stablize the enzyme or enzymes con- which p-aminobenzoic acid is a nutritional requirement cannot
cerned in this system, any possible cofactor requirements cannot effectively utilize ABG as a substitute (20) ; the present authors
be determined. have tested two p-aminobenzoic acid-requiring mutants of E. coli
and have found that neither can use ABG. Also the fact that
DISCUSSION
S. fuecalis will utilize pteroic acid or its reduced forms and not
The data which have been presented in the present paper sug- p-aminobenozic acid or ABG in place of folic acid suggests that a
gest that the biosynthesis of folic acid compounds proceeds (as pteroate compound is an intermediate in folate synthesis.
shown in Fig. 8) by the reaction of hydrosymethyldihydro- The requirements for ATP and Mg++ for the enzymatic syn-
pteridine with p-aminobenzoic acid to give dihydropteroate, thesis of dihydropteroate suggest that a phosphorylated com-
which is then converted, in the presence of glutamate, to di- pound may be an intermediate. A good candidate would be a
hydrofolic acid. The position of hydroxymethyldihydropteri- pyrophosphate ester of hydroxymethyldihydropteridine. This
dine as the most direct pteridine precursor is based on the evi- compound would be expected to be quite reactive since it
dence (a) that, dihydropteroate is the sole product formed from contains an ally1 pyrophosphate-like structure. Similar com-
p-aminobenzoic acid and dihydrofolate is the sole product formed pounds are known to be intermediates for carbon-to-carbon bond
from .4BG and (6) in rate studies osidized pteridines were not as formation (21, 22). Also, a similar pyrophosphate ester, the
effective as substrates as were reduced pteridines. Formulation ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine (which
of the pathway with dihydropteroate as an intermediate rather also contains an ally1 pyrophosphate-like structure) has been
than ABG is based primarily on the evidence, presented in this shown to be an intermediate in the biosynthesis of thiamine (23).
paper, that p-aminobenzoic acid is a much more effective substrate In support of the position of hydrosymethyldihydropteridine
for synthesis of dihydropteroate than is ABG for the synthesis of pyrophosphate as an intermediate is a recent note by Jaenicke
dihydrofolate and also that an enzyme has been discovered in E. and Chan (8) in which they reported that the pyrophosphate of
coli extracts that catalyzes the conversion of dihydropteroate hydroxymethyldihydropteridine can be utilized for folate syn-
and glutamate to dihydrofolate. Many attempts to show that thesis in the absence of ATP.
estracts of E. coli catalyze the formation of ABG from p-amino- Owing to the lability of the enzyme (or enzymes) that converts
benzoic acid and glutamate have met with failures. Other dihydropteroate to dihydrofolate, not much is yet known about
evidence in support of the position of dihydropteroate as an this system. Experiments have been performed that indicate
intermediate instead of ABG is the fact that most organisms for that the system does not form ABG from p-aminobenzoic acid
2542 Biosynthesis of Folk Acid. I Vol. 236, No. 9

and glutamate. Since there is an increasing volume of evidence SUMMARY

that the naturally occurring coenzyme form of folic acid is a Cell-free extracts of Escherichiu coli were active in catalyzing
triglutamate (24), it is possible that the E. coli enzyme system
the conversion of either p-aminobenzoate or p-aminobenzoyl-
could utilize a tripeptide consisting of three glutamate residues glutamate and certain pteridines to compounds with folic acid
as substrate in place of glutamate to yield the triglutamate of activity for Streptococcus jaecalis. When p-aminobenzoic acid
dihydropteroic directly. This tripeptide has not yet been tested was added as substrate, dihydropteroate was formed as product,
as substrate. whereas the use of p-aminobenzoylglutamate as substrate re-
The enzymatic transformation of formylpteridine into a sulted in the formation of dihydrofolate. In this system p-
pteridine which can be used directly for synthesis of dihydro- aminobenzoic acid was several times more active as substrate
pteroate clearly requires FAD, TPN, and DPNH (or DPN and than p-aminobenzoylglutamate.
DPNH-generating system). If hydroxymethyl dihydropteridine Pteridine compounds which were used effectively by the
is the most direct pteridine precursqr, then two types of reduc- enzyme system as precursors of either dihydropteroate or dihy-
tions must occur to convert formylpteridine to this compound: drofolate were 2-amino-4-hydroxy-6-formylpteridine, 2-amino-4-
(a) the reduction of the pteridine ring and (b) the reduction of the hydroxy-6-formyltetrahydropteridine, and 2-amino-4-hydroxy-
formyl group to a hydroxymethyl group. No conclusive evi- 6-hydroxymethyltetrahydropteridine. Adenosine triphosphate
dence is available at present to indicate which cofactors are and Mg++ were necessary components of the system, irrespective
involved in each of these reductive steps. Since TPN is required of which pteridine compound was used as substrate. Reduced
for maximal synthesis when the tetrahydropteridines are used as diphosphopyridine nucleotide, triphosphopyridine nucleotide,
substrate, it seems reasonable to conclude that TPN is perhaps flavin adenine nucleotide, thiamine pyrophosphate, and pyruvate
necessary for the oxidation of the tetrahydropteridine to the were also necessary for maximal synthesis when the B-formyl-

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dihydro compound. The role of TPN when the added substrate pteridine compound was used as substrate; however, only tri-
is formylpteridine is not clear. phosphopyridine nucleotide was required in the presence of
The finding that formylpteridine and hydroxymethyltetra- either of the two active tetrahydropteridines.
hydropteridine are active substrates whereas hydroxymethyl- Dialyzed extracts of E. coli also were able to catalyze a gluta-
pteridine is relatively inactive indicates that the enzyme system mate-dependent reaction whereby reduced forms of pteroic acid
can reduce the pteridine ring structure of formylpteridine but were converted to folate compounds. Evidence was presented
cannot effectively reduce hydroxymethylpteridine. It has also which indicated that the reaction involved the formation
been found3 that neither folic acid nor pteroic acid can be reduced directly of dihydrofolate from dihydropteroate rather than
by the E. coli enzyme system. Thus, the reductive enzyme tetrahydrofolate from tetrahydropteroate.
system seems to be rather specific for formylpteridine. From a consideration of the data presented, the following path-
The data presented in the present paper are in agreement with way for the biosynthesis of dihydrofolic acid was proposed:
those of Shiota (4), who used extracts of L. arabimsus to show
that formylpteridine, hydroxymethylpteridine, and tetrahydro (a) 2-Amino-4-hyd roxy-G-hydroxymethyldihydropteridine +
forms of these compounds could be converted to either pteroic Mg++
acid or folic acid, depending on whether p-aminobenzoic acid or adenosine triphosphate + p-aminobenzoic acid -
ABC was used as substrate. Shiota had to add ATP to his dihydropteroic acid
system but not FAD or pyridine nucleotides. Since he made no (b) Dihydropteroic acid + glutamate -+ dihydrofolic acid
special effort to remove the latter cofactors from his enzyme
preparation, very probably there was enough of these compounds Possible cofactor requirements for Reaction b have yet to be
present so that none had to be added. Shiota did not compare determined.
the relative effectiveness of p-aminobenzoic acid and ABG as
Acknowledgment-It is a pleasure to acknowledge the skillful
substrates, but Jaenicke and Chan (8) reported that less “folate” technical assistance of Mrs. Chai Ho Lo.
was synthesized from p-aminobenzoic acid than from ABG with
E. coli extracts. The latter workers, however, added such large REFERENCES
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the results of the present paper have indicated that excessive LUDWIG, G., SYNNATSCHKE, G., AND WEITKAMP, H., Ab-
amounts of p-aminobenzoic acid are inhibitory. The folate- stracts, IVth Inlernational Congress of Biochemistry, Vienna,
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seems to be quite different from that of E. coli and L. arabinosus. 3. WEYGAND, F., MOLLER, E. F., AND WACKER, A., 2. Naturfors.,
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sac since enzyme systems exist, both in animals and in certain Fifth International Congress on Nutrition, Sept. l-Y, 1960,
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 The Biosynthesis of Folic Acid: I. SUBSTRATE AND COFACTOR
REQUIREMENTS FOR ENZYMATIC SYNTHESIS BY CELL-FREE
EXTRACTS OF ESCHERICHIA COLI
Gene M. Brown, Robert A. Weisman and Donna A. Molnar
J. Biol. Chem. 1961, 236:2534-2543.

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