(Received for publication, October 12, 1994; and in revised form, November 28, 1994)
From the
The 5-methylthio-D-ribose moiety of
5`-(methylthio)-adenosine is converted to methionine in a wide variety
of organisms. 1,2-Dihydroxy-3-keto-5-methylthiopentene anion (an
aci-reductone) is an advanced intermediate in the methionine salvage
pathway present in the Gram-negative bacterium Klebsiella
pneumoniae and rat liver. This metabolite is oxidized
spontaneously in air to formate and 2-keto-4-methylthiobutyric acid
(the -keto acid precursor of methionine). Previously, we had
purified an enzyme (E2) from Klebsiella which
catalyzes the oxidative degradation of the aci-reductone to formate,
CO, and methylthiopropionic acid. To further characterize the reactions
of the aci-reductone we used its desthio analog,
1-2-dihydroxy-3-ketohexene anion (III), which was described
previously. This molecule undergoes the analogous enzymatic and
non-enzymatic reactions of the natural substrate, namely the formation
of formate, CO, and butyrate from III. Experiments with
O
show that E2 is a dioxygenase which
incorporates one molecule of
O into formate and butyric
acid. No cofactor has been identified. We were unable to find an enzyme
which catalyzes the conversion of
1,2-dihydroxy-3-keto-5-methylthiopentane to a keto acid precursor of
methionine. The keto acid is probably produced non-enzymically in Klebsiella. We have, however, identified and purified an
enzyme (E3) from rat liver, which catalyzes the formation of
formate and 2-oxopentanoic acid from III. This enzyme has a monomeric
molecular mass of 28,000 daltons, and no chromophoric cofactor has been
identified. Experiments with
O
show that E3 is a dioxygenase which incorporates an
O
molecule into formate and the
-keto acid. In rat liver CO
formation was not detected.
5`-Methylthioadenosine (MTA), ()a metabolite derived
from S-adenosylmethionine, is converted to methionine in a
wide variety of organisms ((1) , and references therein) The
pathway by which methionine is recycled in the Gram-negative bacterium Klebsiella pneumoniae, as well in rat liver, has been
investigated in our laboratory and elsewhere. In the first step of this
conversion, methythioadenosine 1 (Fig. S1) is hydrolyzed
by a specific nucleosidase to methylthioribose 2 and
adenine(2, 3) . Methythioribose is phosphorylated at
the expense of ATP by a specific kinase to produce
1-phosphomethylthioribose 3 as the
-anomer(4, 5) (bacteria and mammals). This glycosyl phosphate is then
isomerized by aldose-ketose isomerase to 1-phospho-methylthioribulose 4. Ketose phosphate 4 is dehydrated to give (following
ketonization) 2,3-diketo-5-methylthio-1-phosphopentane 5(6) .
Scheme 1: Scheme 1The conversion of methylthioadenosine to methionine.
The metabolism of the diketone 5 is
intriguing. In order for its transformation to methionine to be
complete, this molecule must undergo an oxidative carbon-carbon bond
cleavage to the -keto acid 8, which is then transaminated
to methionine. We have isolated and characterized a bifunctional
enzyme, designated E1, from K. pneumoniae, which
first enolizes the diketone to the phosphoene-diol 6, then
dephosphorylates this compound to the aci-reductone 7(1) . In K. pneumoniae this metabolite
represents a branch point in the methionine salvage pathway, in that it
can be converted to 8 and formate, or to CO, formate and
methylthiopropionic acid(7) .
The work described here was undertaken in order to characterize the enzyme(s) in K. pneumoniae involved in the metabolism of 7. For comparison the metabolism of 7 in rat liver was also investigated.
Scheme 2: Scheme 2The reactions of compounds I and III.
E2 was
concentrated to 40 µM in 20 mM Tris-HCl, pH
7.0, and submitted for electrospray mass spectrometry. Protein under
native conditions 1:1 buffer, 0.2% acetic acid, gave a mass ion of
20,196 ± 10. Protein run under denaturing conditions, 1:1
buffer, 50% acetonitrile, 5% acetic acid, gave identical mass ion.
Atomic absorption spectroscopy was performed to look for iron and zinc. Metal determinations were performed by electrothermal atomic absorption spectroscopy with a Perkin-Elmer model 4100 ZL instrument. Protein, prepared as describe for electrospray mass spectrometry, was diluted in 0.2% metal-free nitric acid (Baker). Aliquots (10 µl) of sample were loaded into pyroltically coated graphite tubes with an AS-70 automatic sampling device. The metal and zinc content is less than 0.01 g atom/mol of enzyme.
To further characterize the reaction,
[C-1-H]III (specific activity,
75,000 cpm/µmol) was prepared(7) . E2 was added to isotopic III under standard assay conditions. Examination of the reaction
mixture by HPLC showed 50% of added radioactivity as formate, 40% in
H
O, and 5% as an unidentified compound. Formate isolated by
HPLC had a specific activity of 78,000 cpm/mol. (
)No
radioactivity was found in butyric acid. There results establishes that
formate is formed primarily, possibly exclusively, from C-1 of III.
The stoichiometry of the reaction requires formation of
another ``1-C'' compound. It is likely that this compound is
derived from C-2 of III. To identify this compound we
synthesized [2-C]III (specific activity,
30,000 cpm/µmol). Upon the action of enzyme on
[2-
C]III no radioactivity was found in
the quenched reaction mixture. We concluded that the compound derived
from C-2 of III is a volatile, water insoluble compound. We have
identified this molecule by its reaction with deoxyhemoglobin as carbon
monoxide (7) . The reaction was carried out under standard
assay conditions in a gas-tight cuvette. Upon completion of the
reaction deoxyhemoglobin was added with a syringe, and the mixture was
scanned between 500-650 nm and 370-450 nm. The absorption
maximum of deoxyhemoglobin at 555 nm disappeared, and new absorption
maxima at 569 and 539 nm appeared. The Soret band shifted from 430 to
419 nm (Fig. 1, A and B). These data indicate
the binding of carbon monoxide to deoxyhemoglobin(10) . The
absorption spectrum for carbomonoxyhemoglobin remains until the ratio
of deoxyhemoglobin to compound III exceeds one, at which point
the deoxyhemoglobin spectrum begins to emerge as dominant.
Concentration of the reaction mixture, followed by gel filtration
chromatography determined that the
C-moiety is associated
with hemoglobin. This result establishes C-2 of III as the
source of CO, and that C-2 is not converted to any other reaction
products. It is concluded that E2 catalyzes as shown in Fig. R1.
Figure 1:
Identification of CO. The reaction
mixture, in an air tight cuvette, in a total volume of 0.5 ml: 0.15
mM III, 0.5 mM MgSO, 50 mM KP
, pH 7.4. E2 (4.8
10
units) was added with a syringe through a septum. The reaction
was monitored at 305 nm, the absorbance maximum of III. After the
spectrum returned to base line, 11 µM deoxyhemoglobin was
added. The spectrum of the reaction mixture was scanned before and
after the addition of deoxyhemoglobin. A, spectrum between 500
and 650 nm; B, spectrum between 370 and 450 nm. (-
-) deoxyhemoglobin;(- - -) deoxyhemoglobin
added to the reaction mixture.
Figure R1: Reaction 1.
Figure R2: Reaction 2.
The studies described above were done with compound III, an analog of 1,2-dihydroxy-5-methylthiopentene anion (7, Fig. S1). We carried out experiments to determine
whether the natural metabolite is a substrate for E2, and E3. Compound 7 (80 nmol) was enzymatically prepared as
described previously (1) and was added to 40 µg of E2 in an oxygen electrode under our standard assay conditions.
Total O consumption was 75 nmol; 68 nmol of
methylthiopropionic acid and and 67 nmol of formate were produced. A
parallel experiment examined the action of E3 on compound 7. O
consumption was 73 nmol, 60 nmol of
-keto
acid, and 68 nmol of formate were generated. These data sugest that the
natural metabolite, 1,2-dihydroxy-5-methylthiopentene anion, is a
substrate for both E2 and E3.
In the methionine salvage pathway S-methylthioadenosine (MTA) is converted to methionine. We had
previously reported that MTA is converted to
2,3-diketo-5-methylthio-1-phosphopentane 5 which, in turn, is
converted to the aci-reductone 7 (Fig. S1). The final
conversion of 7 to the -keto acid precursor of methionine
probably occurs non-enzymically in K. pneumoniae. We have also
purified an enzyme from K. pneumoniae which converts 7 to CO, formate, and
-S-methylthiopropionic acid. All
enzymes and intermediates of the methionine salvage pathway in K.
pneumoniae have now been identified.
In rat liver MTA is also
converted to methionine, it has been established that 5 is a
likely metabolite in the conversion of MTA to methionine(12) .
We have isolated and purified an enzyme from rat liver which converts 5
to the -keto acid precursor of methionine. No corresponding enzyme
is found in K. pneumoniae. The results reported here strongly
suggest that the methionine salvage pathway in rat liver involves
similar reactions as in K. pneumoniae. In the course of this
work we have isolated two dioxygenases E2 and E3.
Some information concerning their mechanism of action has been
obtained.
Carbon monoxide forming enzymes are rare. To date the only mammalian carbon monoxide-forming enzyme is heme oxygenase, which is operative in the pathway of heme degradation(13) . This enzyme acts on protohemin IX to form biliverdin and carbon monoxide. Three other carbon monoxide-forming enzymes have been identified in the fungus Aspergillus flavus and in the bacteria Pseudomonas putida and Arthrobacter sp. Ru61a. These enzymes are dioxygenases which are involved in heterocyclic ring cleavage reactions (14, 15, 16) .
Quercertinase from A.
flavus is a copper-containing dioxygenase which oxidatively
cleaves the ring of quercertin to yield carbon monoxide and depside
(2-protocatechuoylphloroglucinolcarboxylic acid). When O
is used as the dioxygen source, the enzyme
incorporates one
O molecule into the 2 and 4 positions of
the depside, but not into carbon monoxide. When
[3-
C]quercertin was used as a substrate, all of
the radioactivity was present in the carbon of carbon
monoxide(17) . The product distribution of the carbon
monoxide-forming enzyme from K. pneumoniae (E2) is
analogous to that of quecertinase. Recently studies of the CO-forming
dioxygenase from the bacterium Arthrobacter sp. Ru61a have
indicated an analogous reaction to that of E2, in which no
metal or chromophoric cofactor has been identified(18) .
The
reaction of E2 represents a 1,3-oxygenolytic attack which
results in the oxidative cleavage of two carbon-carbon bonds. A
proposed mechanism for the formation of carbon monoxide from compound III is described in Fig. 2. This mechanism necessitates
the formation of a five membered cyclic peroxide intermediate, which
then decomposes to form products. Fig. 2represents the addition
of oxygen to the carbanion tautomer of the aci-reductone to form the
hydroperoxide anion which then attacks the C-3 carbonyl. The reactions
of carbanions with molecular oxygen have been well
studied(19) . An investigation of the autoxidation of ketone
and ester anions by Gersmann and Bickel (20) have shown that
the primary products of these reactions are -hydroperoxides, which
can be isolated in high yields. The formation of the peroxide
intermediate has also been proposed by Abell and Schloss (21) as an intermediate in oxygenase side reactions of
carbanion forming enzymes. They determined that the expression of
oxygenase activity was dependent on the accessibility of the
carbanionic intermediate to molecular oxygen and on the ability of the
enzyme to stabilize the initially formed peroxide anion through
protonation with an enzymic group or through metal coordination.
Figure 2: Proposed mechanism of E2.
There is some reluctance, however, to accept the simple one-step mechanism for the formation of the hydroperoxy anion from the reaction of a carbanion with molecular oxygen, as it involves a violation of the spin conservation rule(22) . Instead, a stepwise single electron transfer from the carbanion to molecular oxygen could be proposed for the formation of the hydroperoxy anion intermediate. Carbon monoxide is a readily extruded functional group in many reactions and its extrusion from the cyclic peroxide intermediate may proceed by a radical, or a concerted mechanism(23, 24) .
E3 is a dioxygenase
which also utilizes the aci-reductone as a substrate. E3,
however, directs a 1,2-oxygenolytic attack on the substrate. A
speculative reaction mechanism is depicted in Fig. 3. This
mechanism suggests the formation of a dioxetane as an intermediate
which subsequently decomposes to form products. The reaction to form
the alkyperoxide intermediate from the attack of the carbanion on
O is the same as that described for E2. The
reaction differs from that of E2 in that the alkyperoxide
anion intermediate adds to C-2 to form the dioxetane, rather than C-3
to form the five-membered cyclic peroxide. The fact that this enzyme,
as well as E2, is not inhibited by catalase suggests that an
alkylperoxide intermediate is not released into solution.
Figure 3: Proposed mechanism of E3.
The metabolism of the aci-reductone 7 has been
investigated in both the K. pneumoniae and rat liver. This
compound is metabolized enzymatically in rat liver by a dioxygenase to
the -keto acid precursor of methionine. This enzyme represents the
final enzymatic transformation of the methionine salvage pathway which
needed to be identified. In K. pneumoniae this metabolite
represents a branch point in the pathway, whose metabolism can follow
two possible routes. This molecule can either undergo a non-enzymatic
oxidation to the
-keto acid precursor of methionine, or can be
acted upon by the CO-forming enzyme to yield CO, formate, and
methylthiopropionic acid. Recent experiments in mammals, which
establish CO as a neurotransmitter(25) , invite interesting
speculation of its possible role as diffusible messenger in bacteria.
Figure R3: Reaction 3.