C O MMU N I C A T I O N S
Table 1. Reaction of AminoF6P in the Presence of Transketolase,
DAHP Synthase, and AminoDAHP Synthase
D-ribose 5-phosphate in A. mediterranei cell-free extract provided
aminoDAHP after purification giving M + 3, M + 2, and M + 1
ions with relative intensities of 10.24%, 0.5%, and 0.42%. On the
basis of this observed retention of both 15N and 2H labeling,
3-amino-3-deoxy-D-fructose 6-phosphate is apparently serving as
a sequestered form of 1-deoxy-1-imino-D-erythrose 4-phosphate and
is not merely a transaminase source of nitrogen.
Enzyme-catalyzed fragmentation of 3-amino-3-deoxy-D-fructose
6-phosphate and enzyme-catalyzed trapping of the resulting 1-deoxy-
1-imino-D-erythrose 4-phosphate to form aminoDAHP has led to
the identification of a defining metabolite in the aminoshikimate
pathway. These observations add to our understanding of the
aminoshikimate pathway as well as raise new questions. Does
1-deoxy-1-imino-D-erythrose 4-phosphate partition between forma-
tion of aminoDAHP and hydrolysis to D-erythrose 4-phosphate and
formation of DAHP in intact A. mediterranei? Complexation of
transketolase with aminoDAHP synthase, which might facilitate
channeling6 of 1-deoxy-1-imino-D-erythrose 4-phosphate from its
formation of 3-amino-3-deoxy-D-fructose 6-phosphate to its con-
densation with phosphoenolpyruvate, remains to be explored. Also,
how is 3-amino-3-deoxy-D-fructose 6-phosphate biosynthesized?
Attention now turns to tracing the steps by which the nitrogen atom
of the aminoshikimate pathway is derived from ammonium ion.
entry
reaction condition
productsc (% yield)d
1
aminoF6P, R5P, PEP;
DAHP (53)
E. coli TktA transketolase (9 unitsa),
E. coli AroFFBR DAHP synthase
(660 unitsb), pH 7.3
2
3
4
aminoF6P, R5P, PEP;
aminoDAHP (2 ( 0.3);
E. coli TktA transketolase (9 unitsa),
A. mediterranei RifH aminoDAHP
synthase (64 unitsb), pH 7.3
aminoF6P, R5P, PEP;
DAHP (35)
aminoDAHP (7 ( 0.2);
DAHP (19); AHBA (3);
Tyr (5); Phe (5)
A. mediterranei cell-free extract
(DAHP synthase activity of 0.2
unitsb), pH 7.3
F6P, R5P, PEP, glutamine,
(NH4)2SO4; A. mediterranei
cell-free extract (DAHP synthase
activity of 0.2 unitsb), pH 7.3
DAHP (29)
a Transketolase was assayed according to ref 5a. b AminoDAHP synthase
was assayed as DAHP synthase activity according to ref 5a. c See the legend
to Scheme 1 for abbreviations. d Yields are 1H NMR yields of aminoDAHP,
DAHP, and AHBA purified to homogeneity and of L-tyrosine and L-phen-
ylalanine purified to a binary mixture. Response factors and quantification
of product concentrations were based on integration relative to 3-(trimeth-
ylsilyl)propionate-2,2,3,3-d4.
Acknowledgment. Professor Heinz G. Floss provided rifH as
well as numerous unpublished observations and hypotheses regard-
ing the aminoshikimate pathway. Research was supported by a
contract from F. Hoffmann-La Roche Ltd.
and location in the rif biosynthetic gene cluster.2c,d 3-Amino-3-
deoxy-D-fructose 6-phosphate, D-ribose 5-phosphate, and phospho-
enolpyruvate reacted in the presence of TktA and RifH to form
DAHP along with a 2% yield of aminoDAHP (entry 2, Table 1).
Addition of 3-amino-3-deoxy-D-fructose 6-phosphate, D-ribose
5-phosphate, and phosphoenolpyruvate to crude cell lysate prepared
from A. mediterranei (ATCC 21789) afforded a 7% yield of
aminoDAHP (entry 3, Table 1) along with formation of DAHP,
AHBA, L-tyrosine, and L-phenylalanine. Far fewer units of DAHP
synthase activity were present in A. mediterranei cell-free lysate
(entry 3, Table 1) relative to the units of RifH activity (assayed as
a DAHP synthase) employed with TktA transketolase (entry 2,
Table 1). As a control experiment, D-fructose 6-phosphate, D-ribose
5-phosphate, phosphoenolpyruvate, and glutamine and (NH4)2SO4
as possible sources of nitrogen were incubated in crude A.
mediterranei cell-free extract (entry 4, Table 1). No aminoDAHP
formation was detected.
The possibility remained that 3-amino-3-deoxy-D-fructose 6-phos-
phate might undergo a transamination reaction thereby being the
source of the nitrogen atom but not of 1-deoxy-1-imino-D-erythrose
4-phosphate in its entirety. To address this possibility, 3-[15N]-
amino-3-deoxy-D-6,6-[2H2]-fructose 6-phosphate was synthesized
from D-6,6-[2H2]-fructose and 15NH2OH‚HCl via the synthetic route
specified in Scheme 1. Dilution with unlabeled 3-amino-3-deoxy-
D-fructose 6-phosphate afforded material that gave M + 3, M + 2,
and M + 1 ions with relative intensities of 10.97%, 0.24%, and
-0.6%, respectively, when analyzed by negative ion electrospray
mass spectrometry. Incubation with phosphoenolpyruvate and
Supporting Information Available: Synthesis of aminoF6P and
3-[15N]-6,6-[2H2]-aminoF6P and enzymatic formation of aminoDAHP
and 3-[15N]-6,6-[2H2]-aminoDAHP (PDF). This material is available
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