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INTRODUCTION |
Escherichia coli mutants, which are unable to express
both the iron- and the manganese-containing superoxide dismutases,
exhibit several phenotypic deficits, among which are
oxygen-dependent auxotrophies for branched chain,
sulfur-containing (1), and aromatic amino acids (2). The requirement
for branched chain amino acids was explained on the basis of the
oxidative inactivation of the dihydroxy acid dehydratase (3-6), which
catalyzes the penultimate step in the relevant biosynthetic pathway.
The requirement for sulfur-containing amino acids was attributable to
leakage of sulfite from the cells (7, 8).
We have now investigated the aromatic amino acid auxotrophy of the
sodA sodB strain and find an explanation quite
different from those that rationalized the other amino acid
auxotrophies. Thus, the aromatic biosynthetic pathway begins with the
condensation of erythrose-4-phosphate
(E-4-P)1 with phosphoenol
pyruvate (PEP) to yield
3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), and the
production of E-4-P in turn is dependent on the sequential actions of
transketolase (TK) and transaldolase. It has been shown that the
intermediate of the TK reaction, which is 1,2-dihydroxyethyl thiamine
pyrophosphate, is oxidized by O2
(9, 10). We now
report that this oxidation interferes with the production of E-4-P and
thus accounts for the decrease of aromatic biosynthesis in aerobic
sodA sodB E. coli.
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MATERIALS AND METHODS |
The E. coli strains used were AB1157 parental: JI132
(11), sodA sodB; BJ502 (Hfr C str-stkt-2) (12,
13), which lacks the ability to produce transketolase A; and
BJ502tkt+, which has a functional tkA and
DH52/pkD44B, prepared by insertion of a 5-kb BamHI fragment
encoding transketolase A into the TcR cosmid pLAFR3 (14).
These stains have TK activities of 0.03, 0.17, and 1.5 units/mg,
respectively (15).
Unless otherwise specified all strains were grown overnight in aerobic
LB medium at 37 °C and were then diluted into the test media. When
the test medium was one that contained 17 amino acids, but lacking Tyr,
Trp, and Phe, the inoculum was taken from an overnight culture in
anaerobic minimal medium to avoid transfer of aromatic amino acids from
LB medium. The 17-amino acid medium contained M9 salts (16)
supplemented with 100 mg/liter concentrations of the 20 amino acids
commonly found in proteins except the three aromatics. In each case the
defined medium also contained 0.2% glucose plus 3 mg/liter pantothenic
acid and thiamine. Minimal medium contained the vitamins mentioned
above plus 100 mg/liter Thr, Leu, His, Pro, and Arg and 0.2% glucose.
When enzyme activities were to be assayed, cultures were grown to
A600 nm of 0.5-0.6 and were washed twice in
the chilled buffer to be used in the assay. The washed cells were
disrupted in a French press, and the extracts were clarified by
centrifugation before assay. The assays were performed as described in
the published reports: thus DAHP synthase (17), 3-dehydroquinate
synthase (18), 3-dehydroquinate dehydratase (19), shikimate
dehydrogenase (20), TK (12, 21), and transaldolase (21).
The following substrates, needed for these assays, were prepared by
published procedures as follows: DAHP (22), 3-dehydroquinate (23), and
5-dehydroshikimate (24). Metabolites were assayed in the cell extracts
as follows: PEP (25), ATP (Sigma bioluminescent assay kit) and E-4-P
(26). Culture fluids that had been centrifuged and then passed through
a 0.22-µm filter were assayed for DAHP (27) and 3-dehydroquinate (27)
to check for leakage of these intermediates. Hyperoxia was achieved in
specifically fabricated stainless steel vessels, which were maintained
at 37 °C after pressurization.
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RESULTS |
Shikimate Relieves the Aromatic Amino Acid Auxotrophy Imposed by
Lack of SodA and SodB--
As previously noted (2) the sodA
sodB strain of E. coli exhibited an auxotrophy
for Phe + Tyr + Trp. This auxotrophy is not attributable to leakage of
metabolic intermediates, because it was not relieved by 0.25 M sucrose, which did relieve the Cys + Met auxotrophy (7,
8) (data not shown). To localize the point of blockage of aromatic
biosynthesis, shikimate was tested and was found to replace the
aromatic amino acid requirement. These results are shown in Fig.
1. Thus line 5 illustrates the very slow growth seen in the absence of the aromatics compared with the
rapid growth when these were present (Fig. 1, line 1). Shikimate allowed growth almost as well as did the aromatics (Fig. 1,
line 2). The shikimate was not a nonspecific stimulator of growth, because it did not augment the slow growth in the absence of
Lys, Ser, and Gly (Fig. 1, lines 3 and 4).

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Fig. 1.
Shikimate substitutes for the aromatic amino
acids in the sodA sodB strain. JI132 was grown
overnight in anaerobic minimal medium and was then diluted 200-fold
into the defined test media, which contained: line 1, all 20 amino acids; line 2, all but the aromatic amino acids, plus
1 mM shikimate; line 3, all amino acids except
Lys, Ser, and Gly plus 1 mM shikimate; line 4,
all amino acids except Lys, Ser, and Gly; and line 5, all
but the aromatic amino acids.
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Enzyme Activities--
There are four enzymes on the pathway
to the aromatic amino acids that precede shikimate; these are
DAHP-synthase, 3-dehydroquinate synthase, 5-dehydroquinate dehydratase,
and shikimate dehydrogenase. These were assayed in extracts of the
parental and the sodA sodB strains using
published procedures (12, 17-21) and preparing the necessary
substrates as described (22-24). None of these activities was found to
be much lower in the sodA sodB extracts than in
the parental strain when the extracts were assayed promptly (data not
shown). It appeared that none of these four enzymes is a target for
O2
and therefore that the effect of
O2
on aromatic biosynthesis was exerted at some still
earlier point. Because the first step in this pathway involves the
condensation of PEP with E-4-P, the extracts were assayed for PEP and
ATP. The results showed that although ATP > PEP, there was no
significant difference between the sodA sodB and
parental strains (data not shown). These negative results focussed our
attention on E-4-P. E-4-P was not detectable in extracts of either
AB1157 or JI132.
Transketolase--
E-4-P is made by the action of transaldolase on
sedoheptulose-7-phosphate plus glyceraldehyde-3-phosphate, and
sedoheptulose-7-phosphate in turn is made by the action of TK on
D-xylulose-5-phosphate plus
D-ribose-5-phosphate. TK is thus essential for the
production of E-4-P and was considered a likely target for
O2
both because of the previously reported oxidation
of its 1,2-dihydroxyethyl thiamine pyrophosphate intermediate by
O2
(9, 10) and because TK-deficient mutants of
E. coli have been reported (12) to be aromatic amino acid auxotrophs.
If O2
was interfering with the TK reaction, then the
sodA sodB strain should have difficulty using
ribose as a carbon source (12), because ribose catabolism depends on
the direct oxidative pathway that uses TK. Fig.
2 shows that the parental strain grew somewhat better on glucose than on ribose (compare lines 1 and 2), whereas the sodA sodB strain
grew very much better on glucose than on ribose (lines 3 and
4). This result is in accord with expectations and with the
reports that TK mutants do not grow on pentoses (12).

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Fig. 2.
The sodA sodB strain exhibits a
relative inability to grow on ribose. Overnight cultures of JI132
or of AB1157 in anaerobic minimal media were diluted 200-fold into
media lacking the aromatic amino acids containing 2.5 mg/ml shikimate
and 0.2% glucose or ribose. Line 1, AB1157 in glucose;
line 2, AB1157 in ribose; line 3, JI132 in
glucose; line 4, JI132 in ribose.
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The ability of transketolase to scavenge O2
in a
manner dependent on its keto sugar phosphate substrates has been
reported (9, 10) and has been attributed to a rapid oxidation of the 1,2-dihydroxyethyl thiamine pyrophosphate intermediate by
O2
. In full accord with these reports we found that
TK caused a dose-dependent inhibition of the reduction of
cytochrome c by the aerobic xanthine oxidase reaction and
that this was entirely dependent on the presence of either
fructose-6-phosphate or xylulose-5-phosphate (data not shown).
O2
Inactivates Transketolase--
The first step
in the oxidation by O2
of the thiamine-bound glycol
intermediate of the TK must be a univalent process yielding a bound
glycol radical. The final production of glycolate, shown earlier (9,
10), must depend on additional steps. It appeared possible that the
bound glycolate radical might, with some frequency, oxidize and
inactivate the enzyme. In that case TK activity should be lower in
the sodA sodB strain than in the parental strain. As shown in Fig. 3, TK activity was lower
in the SOD null strain than it was in the parental strain. This was
also true, but to a lesser degree, of transaldolase, whereas there were
no differences in glucose-6-phosphate dehydrogenase and
6-phosphogluconate dehydrogenase.

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Fig. 3.
TK activity is low in the sodA
sodB strain. JI132 and AB1157 at midlog phase in M9 medium
plus casamino acids were washed, extracted, and assayed for enzymatic
activities as described under "Materials and Methods." The
black bars denote JI132, and the gray bars denote
AB1157. G6PD, glucose-6-phosphate dehydrogenase;
6PGD, 6-phosphogluconate dehydrogenase; TA,
transaldolase.
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Effect of Paraquat--
If the relative paucity of TK in the
sodA sodB strain had been attributable to
inactivation of the enzyme by O2
, then paraquat
should lower TK even in the parental strain. That this was the case is
shown in Fig. 4. Thus line 1 presents the increase in TK activity, which accompanied the outgrowth
of a stationary phase inoculum of AB1157 in LB medium. In contrast, the
increase of TK activity was much less in the case of JI132 (Fig. 4,
line 2). Paraquat at 20 µM suppressed the
increase of TK in the parental strain (Fig. 4, line 3) and
at 4 µM did so in the sodA sodB
strain (Fig. 4, line 4). It should be noted that these low
levels of paraquat did not significantly inhibit growth in this rich
medium.

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Fig. 4.
The growth-related increase in TK activity is
eliminated by paraquat. JI132 and AB1157 from an overnight culture
in LB were diluted 50-fold into fresh LB containing: line 1,
AB1157 in LB; line 2, JI132 in LB; line 3, AB1157
in LB plus 20 µM paraquat; and line 4, JI132
in LB plus 4 µM paraquat.
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Effect of Hyperoxia on Growth--
Paraquat increased
O2
production, but it did so by diversion of electron
flow from useful pathways, and this can complicate interpretation of
its effects. For this reason we tested the effect of hyperoxia,
anticipating that it should inhibit growth more when the carbon source
was ribose than when it was glucose. Fig. 5, line 1, represents the
growth of AB1157 at 5.5 atm of O2 on glucose, whereas
line 2 shows the much slower growth on ribose. It should be
noted that growth under ordinary atmospheric conditions was much less
affected by switching from glucose to ribose (Fig. 2, lines
1 and 2).

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Fig. 5.
Hyperoxia selectively suppresses growth on
ribose. Overnight cultures of AB1157 in LB medium were diluted
200-fold into M9 medium plus casamino acids containing either glucose
(line 1) or ribose (line 2) at 0.2%. Subsequent
growth was under ~5.5 atm of O2.
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Effect of Hyperoxia on TK Activity--
If the relative paucity of
growth on ribose compared with glucose, imposed by hyperoxia, was
attributable to inactivation of TK by O2
, then we
should be able to see this inactivation most clearly when de
novo protein synthesis was inhibited, and we should be able to
prevent the inactivation with a cell-permeant catalyst of the
dismutation of O2
(28). The affirmation of these
expectations is presented in Fig. 6. Thus
bar 4 in Fig. 6A gives the activity of TK in
midlog AB1157 treated with chloramphenicol and kept at 0 °C for
3.5 h. Bar 3 shows the decline in TK, which occurred
when the cells were kept at 37.5 °C in the presence of
chloramphenicol for that period. Bars 1 and 2 illustrate that the extent of TK inactivation under 3.2 atm of
O2 was made apparent only when protein synthesis was inhibited (Fig. 6A, bar 2). Fig. 6B presents the
protection by the SOD mimic MnTM-2-PyP (28). Thus bar set 1 is a control showing that the mimic had no effect on TK activity in
cells kept on ice in the presence of chloramphenicol Bar set
2 shows that 25 µM MnTM-2-PyP diminished the loss of
TK seen in air in the presence of chloramphenicol, whereas bar
set 3 shows the profound inactivation of TK under 3.5 atm of
O2 and the striking protection provided by the SOD mimic.
Hence the inactivation of TK was most likely attributable to
O2
and could be seen even in the SOD-replete strain
when O2
production was increased by raising
pO2.

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Fig. 6.
TK is inactivated by hyperoxia and protected
by the SOD mimic Mn(III)TM-2-PyP. AB1157 was grown to midlog phase
(A600 nm = 0.5-0.6) and was then divided into
several aliquots, some of which were treated with 200 µg/ml
chloramphenicol followed 15 min later by exposure to air or 3.2 atm of
O2 for 3.5 h at 37.5 or 0 °C before washing,
extraction, and assay for TK activity. A, bar 1,
3.2 atm of O2; bar 2, 3.2 atm of O2
plus chloramphenicol; bar 3, air plus chloramphenicol;
bar 4, air plus chloramphenicol at 0 °C. B,
black bars, without the SOD mimic; gray bars,
with 25 µM SOD mimic. Conditions were as in A
except: bar set 1, chloramphenicol at 0 °C; bar set
2, air plus chloramphenicol, 37 °C; and bar set 3,
3.5 atm of O2 plus chloramphenicol.
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Effect of Overproducing TK--
If inactivation of TK by
O2
was limiting growth under hyperoxia, then a strain
overproducing TK ~10-fold, by virtue of a cosmid bearing the TK gene,
should have a growth advantage under hyperoxia. In Fig.
7, comparison of lines 1 and
2 shows that this was the case. It should be noted that
under normoxia the TK overproducer actually grew somewhat slower than
its parental strain.

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Fig. 7.
Overproduction of TK facilitates growth under
hyperoxia in the absence of aromatic amino acids. E. coli
was diluted 200-fold from an overnight culture into fresh medium
lacking Phe, Tyr, and Trp and was incubated under ~5 atm of
O2. Line 1, DH5a/pKD44B; line 2,
Bjtkt+. The strain in line 1 had approximately
nine times higher TK activity than did the strain in line
2.
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Substrate-dependent Inactivation of TK by
O2
--
TK, either as the purified enzyme (Fig.
8A) or in an extract of JI132
(Fig. 8B), was inactivated when exposed to a photochemical flux of O2
in the presence of fructose-6-phosphate
(Fig. 8A, line 3), and this inactivation was diminished by
10 µg/ml SOD (Fig. 8A, line 2) and was dependent on the
glycol-donating substrate (Fig. 8A, line 1). The TK activity
in an extract of JI132 (Fig. 8B) was similarly inactivated
(Fig. 8B, line 3) and was similarly protected by
10 µg/ml SOD (Fig. 8B, line 2). Fig. 8B,
line 1, illustrates the stability of the TK activity in
these extracts in the absence of the photochemical flux of
O2
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Fig. 8.
A photochemical flux of O2
inactivates TK dependent on a glycol donor and prevented by SOD.
Reaction mixtures contained 0.1 mM thiamine pyrophosphate,
3.0 mM fructose 6-phosphate, 11.5 µg of riboflavin, 2.5 µl of tetramethyl ethylenediamine, 2.0 mM
MgSO4, and either purified TK or a crude extract of JI132
in 3.0 ml of 5.0 mM Tris at pH 8.0 and at 25 °C. The
samples in 3.0-ml quartz cuvettes were exposed to light from a pair of
parallel 20-watt fluorescent tubes (daylight) at a distance of ~5 cm
or were kept dark. At intervals samples were taken for assay of TK.
A, with purified TK. Line 1, no
fructose-6-phosphate; line 2, 10 µg/ml Cu, ZnSOD;
line 3, no additions. B, crude extract of JI132.
Line 1, kept dark; line 2, 10 µg/ml Cu, ZnSOD;
line 3, no additions.
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Effect of Iron--
We have previously noted (29) that enrichment
of growth media with iron relieved some of the phenotypic deficits of
the sodA sodB strain. Iron enrichment elevated the levels of
[4Fe-4S] containing dehydratases, and we surmised that the oxidative
inactivation and reductive repair of these [4Fe-4S] clusters was
providing a pathway for the scavenging of O2
and was
thus protecting less reparable targets. The data in Fig. 9 show that TK is one of those less
repairable targets of O2
. It should be noted that
growth in iron-enriched medium did not affect the activities of DAHP
synthase, dehydroquinate synthase, 5-dehydroquinate dehydrogenase, or
shikimate dehydrogenase (data not shown).

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Fig. 9.
Iron supplementation facilitates growth and
protects TK activity in JI132. An overnight culture of JI132 in
anaerobic minimal medium was diluted 200-fold into the defined medium
lacking Tyr, Phe, and Trp with or without 0.5 mM
FeCl2. A, growth with (line 1) and
without (line 2) iron enrichment;
A600 nm caused by to FeCl2 per se
was subtracted from these readings. B, TK activity with
(gray bar) and without (black bar) Fe
enrichment.
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DISCUSSION |
The blockade of aromatic amino acid biosynthesis imposed by
O2
imposes an auxotrophy for these amino acids that
was relieved by shikimate. Hence that blockade was in the early steps
of the aromatic biosynthetic pathway. None of the enzymes catalyzing the early steps in this pathway was found to be O2
sensitive. Because this pathway begins with the condensation of PEP
with E-4-P, and because an intermediate in the TK reaction was already
known to be oxidized by O2
with a rate constant of
~106 M
1 s
1 (10),
it appeared possible that the problem might be imposed by this reaction.
The sodA sodB strain of E. coli did
exhibit a relative deficiency in its ability to use ribose as a carbon
source, whereas the ability of the SOD-replete strain to grow on ribose
could be diminished by raising O2
production through
application of paraquat or hyperoxia. TK activity was low in the
sodA sodB strain, whereas there were no differences in
glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase and only a marginally significant difference in transaldolase. Paraquat
or hyperoxia lowered the TK activity in the bacteria, and a
cell-permeant mimic of SOD activity protected. The inhibition of growth
imposed by hyperoxia was lessened by overproduction of TK, and TK
activity in either purified form or in crude extracts was inactivated
by a photochemical flux of O2
and was protected by SOD.
All of these data indicate that the synthesis of E-4-P was compromised
by the O2
-TK reaction and that lack of E-4-P
prevented the first step of the aromatic pathway. It should be noted
that leakage of intermediates from the cells into the medium was tested
for and was not observed (data not shown).
It is interesting that, although carefully sought after, E-4-P has
never been detected in tissue extracts when specific assay methods were
used (26, 30). The explanation offered for this failure has been the
spontaneous dimerization of this sugar (26, 31). However, that
dimerization has been seen to lower the concentration of E-4-P by only
75% in in vitro tests; so it would not be likely to
entirely prevent detection of this sugar. Moreover a dimerization reaction would proceed even less well when the concentration of the
monomer was very low. We have noted a toxicity of short chain sugars
and have related it to the reactivity of the exposed carbonyl group and
to the formation of enediolate tautomers, which can oxidize to very
toxic
-
diketones (32). Hence short chain sugars should be
kept at vanishingly low concentrations to prevent this. Tamarit
et al. (33) have examined the proteins damaged in E. coli exposed to oxidative stresses. They identified these proteins
by looking for protein carbonyls and did not find transketolase among
them. Possibly transketolase escaped their notice because its oxidative
inactivation does not generate a carbonyl group.