+
+
Biosynthesis of 3-Amino-5-hydroxybenzoic Acid
J. Am. Chem. Soc., Vol. 118, No. 32, 1996 7487
acid (QA),10 or 3-dehydroquinic acid (DHQ)7b into the mC7N
unit were unsuccessful. The nonincorporation of shikimate
pathway intermediates could be due to impermeability of the
cell membranes of the producing organisms to these compounds,
demonstrated for SA in the rifamycin-producer, Amycolatopsis
mediterranei,8c or could indicate that the biosynthesis of the
mC7N unit branches off from the shikimate pathway at an earlier
stage. The former possibility was excluded for the case of
ansatrienin A (cf. Scheme 1) by showing that 13C-labeled SA
is efficiently incorporated into the cyclohexanecarboxylic acid
moiety of this antibiotic, but not into its mC7N unit.11 Through
genetic studies Gygax et al.8c established that the branch point
for formation of the mC7N unit must lie before DHQ in the
shikimate pathway. Their studies showed that a transketolase
inactivated mutant of A. mediterranei could not produce
aromatic amino acids and rifamycin, but a DHQ synthase
inactivated mutant could synthesize rifamycin.
3-Amino-5-hydroxybenzoic acid (AHBA) was shown to be
a specific and proximate precursor of the mC7N unit both by
feeding experiments with labeled AHBA12 and by complemen-
tation experiments with blocked mutants.13 AHBA was ef-
ficiently and specifically incorporated into all the different
ansamycins12a,14-18 and ansamitocins19 and into mitomycins (cf.
Scheme 1).12b However, this still leaves unexplained how
AHBA is formed via the shikimate pathway. While it was first
thought that the nitrogen is attached to C-3 of, e.g., 3-dehy-
droshikimic acid (DHS),5a the analysis of labeling patterns in
mitomycin7b and 13C-13C coupling patterns in geldanamycin,20
naphthomycin18 and ansatrienin21 revealed that the nitrogen is
actually linked to the carbon corresponding to C-5 of shikimic
acid. This led Hornemann et al.7b to suggest aminoDAHP,
thought to arise from 3-deoxy-D-arabino-heptulosonic acid
7-phosphate (DAHP), as the precursor of the mC7N unit,
whereas Rinehart et al.20 proposed transamination of a hypo-
thetical 5-dehydroshikimic acid derived from 3-dehydroshikimic
acid.
with erythrose 4-phosphate (E4P); condensation with phospho-
enolpyruvate (PEP) then gives aminoDAHP directly. Cycliza-
tion and dehydration, either by the normal shikimate pathway
enzymes or by a separate set of enzymes then produces
aminoDHS, which is aromatized to AHBA. In the present paper
we report results of experiments with cell-free extracts of the
rifamycin producer, A. mediterranei, and the ansatrienin pro-
ducer, Streptomyces collinus, which provide considerable sup-
port for this hypothesis. Some of the data have been commu-
nicated in preliminary form.23
Materials and Methods
General Chemicals and Procedures. 1H-NMR spectra were
recorded on an IBM-Bruker AF-300 NMR spectrometer, using D2O
as the solvent. Chemical shifts were determined relative to the residual
proton absorption of D2O as internal standard. All 1H-NMR chemical
shifts are reported as δ values in parts per million (ppm). High
resolution fast atom bombardment mass spectra (FAB-MS) were
obtained in a Fisons Instruments VG 70SEQ spectrometer. GC-MS
analyses were carried out on a Hewlett-Packard 5970A gas chromato-
graph connected to a 5790 mass selective detector.
Unlabeled AHBA and [1-13C]AHBA,24 DAHP,25 DHQ,26 and DHS27
were prepared by previously described chemical or enzymatic proce-
dures. The synthesis of aminoDAHP has been described.28 The N-(2,4-
dinitrophenyl) derivative of aminoDAHP (DNP-aminoDAHP) was
prepared following a procedure employed by Basmadjian and Floss.29
PEP, E4P, QA, SA, phenylmethanesulfonyl fluoride (PMSF), 2,4-
dinitrophenyl fluoride, and silylating reagent (SIGMA-SIL-A) were
purchased from Sigma (St. Louis, MO). [amide-15N]Glutamine and
15NH4Cl (98% 15N) were obtained from Cambridge Isotope Laborato-
ries. [1-14C]PEP (23 mCi/mmol) and sodium [1-14C]pyruvate (23 mCi/
mmol) were obtained from Amersham. [1-14C]DAHP was isolated from
the incubations of A. mediterranei cell-free extracts with [1-14C]PEP
and E4P and purified to radiochemical homogeneity by repeated paper
chromatography.
Microbial Cultures and Fermentations. A. mediterranei strain
S699 was a gift from Professor Giancarlo Lancini (Lepetit Research
Laboratory, Geranzano, Italy) and S. collinus Tu¨1892 from Professor
Axel Zeeck (Institut fu¨r Organische Chemie, Universita¨t Go¨ttingen,
Germany). E. coli AB2834/pIA321, a genetically engineered over-
producer of shikimate dehydrogenase, was kindly provided by Professor
John R. Coggins (Department of Biochemistry, University of Glasgow,
UK) and E. coli RB791/pJB14, a genetically engineered overproducer
of DHQ synthase, by Professor Jeremy R. Knowles (Department of
Chemistry, Harvard University). E. coli AB2834/pIA321,27 E. coli
RB791/pJB14,30 and S. collinus Tu¨189215 were grown as previously
described.
Growth of A. mediterranei S699. A plate culture from a solid
medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, 20
g of agar, and 1 L of distilled water, pH 7.3) was used to inoculate
vegetative medium (5 g of meat extract, 5 g of peptone, 5 g of yeast
extract, 2.5 g of enzyme hydrolysate of casein, 20 g of glucose, 1.5 g
of NaCl, and 1 L of distilled water).31 Vegetative cultures (50 mL of
medium per flask) were grown for 2 days in 500 mL shake flasks with
spring coils at 28 °C and 250 rpm (ISF-4-V shaker, Adolf Kuhner AG).
After the growth was complete, contamination was checked by light
microscopic examination. Four milliliter portions of the vegetative
In both these proposals the nitrogen would originate from
the amino nitrogen of glutamic acid via a transamination
reaction. However, Jiao et al.22 reported that the amide nitrogen
of glutamine was the best source of the nitrogen of rifamycin
B. This and mechanistic considerations led us21a to propose
the pathway shown in Scheme 2 for the formation of AHBA.
The key feature is the suggested operation of a modified DAHP
synthase containing an additional protein subunit or domain
which binds and hydrolyzes glutamine, generating in the active
site a molecule of ammonia. The latter forms a Schiff’s base
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