Download Unusual dehydrations in anaerobic bacteria

FEMS MicrobiologyReviews88 (1992) 211-232
© 1992 Federation of European MicrobiologicalSocieties0168-6445/92/$15.00
Published by Elsevier
211
FEMSRE 00225
Unusual dehydrations in anaerobic bacteria
W o i f g a n g Buekel
Laboratorium f~r Mikrobiologie, Fachbereich Biologie, Philipps-Unicersitiit, Marbur~ FRG
Received27 January 1992
Accepted 6 February 1992
Key words: Hydroxyacyl-CoA dehydratase; L-Serine dehydratase; Riboflavin; F A D ; Iron-sulfur cluster;
ATP
1. S U M M A R Y
In amino acid fermenting anaerobic bacteria a
set of unusual dehydratases is found which use
2-hydroxyacyi-CoA, 4-hydro:~butyryl-CoA or 5hydroxyvaleryl-CoA as suhstratcs. The extremely
oxygen-sensitive 2-hydroxyacyl-CoA dehydratases
catalysing the elimination of water from (R)lactyi-CoA to acryloyl-CoA or from (R)-2-hydroxyglutaryl-CoA to glutaconyI-CoA contain
iron-sulfur clusters as well as riboflavin and require additional activation by ATP. The dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA is
catalysed by a moderately oxygen-sensitive enzyme also containing an iron-sulfur cluster and
FAD. In all these reactions a non-activated C H-bond at C 3 has to be cleaved by mechanisms
not yet elucidated. The dehydration of 5-hydroxyvaleryl-CoA to 4-pentenoyl-CoA, however, has
been characterised as a redox process mediated
by enzyme-bound FAD. Finally, an iron-sulfur
Correspondence to: W. Buckel, Laboratorium fdr Mikrobiologie, Fachbereich Biologie, Philipps-Universit~it,W-3550 Marburg, FRG.
cluster-containing but pyridoxal-phosphate-independent L-serine dehydratase is described.
2. I N T R O D U C T I O N
Dehydration is an ubiquitous reaction in biology. Well-known examples of enzymes catalysing
dehydrations are fumarase and aconitasc in the
citric acid cycle, crotonase in the E-oxidation of
fatty acids, enolase in glycolysis and 6-phosphogluconate dehydratase in the EntncPDoudoroff
pathway. In addition there arc many dehydratases for distinct biosynthetic purposes such as
threonine dchydratase and dihydroxy acid dehydratase in the pathway leading to isoleucine. The
catalogue of enzymes [1] lists about 100 different
dchydratases under the systematic name of carbon-oxygen lyases (EC 4.2). Almost all of these
enzymes are specific for substrates in which the
hydrogen to be eliminated as a proton is activated
by an adjacent, electron withdrawing group as are
carboxylatc, thiolester or ketone, whereas the hydroxyl group (or an alkoxide) to be removed is
located in the/3-position to the activating residue.
Essentially four types of substrates are delay-
212
drated (Fig. 1): (i) /3-hydroxy-a,/3-dicarboxylic
acids like citrate, isocitrate or malate. The dehydration of the latter has been described as anti.
elimination [2] with a carbanion as intermediate
[3]. (ii) /3-HydroxT acids with an additional hydroryl, phospho- or amino group in the a-position like gluconate, 2-phosphoglycerate or serine.
In the case of gluconate the resulting enol tautomerises to 2-oxo-3-deoxygluconate, whereas the
enamine derived from serine tautomerises to the
ketimin followed by hydrolysis to pyruvate. (iii)
$-Hydroxyacyl-CoA esters are dehydrated in asynchronous syn-process [4,5]. Interestingly, no
simple free/3-hydroxy acid has been detected as
substrate for a dehydratase unless converted to
the thiolester. Obviously, the activation of the
a-hydrogen by a carboxylate alone is not sufficient for the dehydration. (iv) Finally, water is
removed from /3-hydroxyketones also in a synelimination, like 5-dehydroquinate being dehydrated to 5-dehydroshikimate [6].
Glu ~
" ~ NAD+ + H20
o
•OOC~ ' %
z[n]/~
/ NADH
N'~.ooNI
~*
AcetyI,CoA
~ H
CoAS'~s~
0
~
H20
CoAS~ . , ~
O
~.~H~
2
CoAS~.~
O
ATP
3 Alanine + 2 H z O 2 ~ 3 NH4+ + Acetate" + 2 Propionate" + CO2
n4r ~,~H,
H0_
H
~,
H
COO"
Fig. 2. Fermentation of alanine by Clostridium propionicum.
H
CO0"
X = OH or NHs÷
R-c~-~=c0o0
H
m
OH O
R
H
$CoA
H
8CoA
ne
H
"O0C
gO
~
0
R
H
"~
HO
Fig. 1. "Usual' dehydrations. The individual reactions are
described in the text.
In recent years, however, dehydratases have
been isolated from anaerobic bacteria with substrates except from the gengralisations mentioned
al?ove. It has been rgalised that several anaerobes
are capable of reducing proteinogenous amino
acids to short-chain fatty acids via the corresponding (R)-2-hydroxyacyl-CoA esters following
dehydration to the enoyI-CoA esters [7]. Examples are the fermentations of alanine by Clostridium propionicum [8] (Fig. 2) and glutamate by
Acidaminococcus fermenmns [7] (Fig. 3). The dehydration of (R)-2-hydroxyacyl-CoA esters, e.g.
(R)-lactyl-CoA to acryloyl-CoA, is of considerable interest because of "a potential mechanistic
novelty of such a reaction" first pointed out by
Anderson and Wood [9]. The hydrogen to be
eliminated at the C 3 position cannot be activated
by the thiolester, and, in the reverse direction,
213
÷H3N H
.ooc~COO
-
~__~.~NAg÷ ~" HIO
NADH , 1 . ~
eneglyco! and glycerol, from which non-activated
hydrogens are removed during catalysis: diol dehydratase from enterobacteria and propionibacteria as well as glycerol dehydratase from enterobacteria. However, both enzymes require adenosylcobalamin ( c o e ~ e
BI2) and therefore belong to a group of enzymes which have been
reviewed already by others several times [11-13]
and are not considered in this paper.
3. 2 - H Y D R O X Y A C Y L - C o A
TASES
Sum:2 Glutamate'+ l I|10 + H+ - - ~ 2 NH4÷ ÷ 2 COl + 2 Actqmle"÷ But~rate"
Fig. 3. Fermentation of glutamate by Acidaminococcusfermenmns (hydroxyglutaratepathway), modified from [54]. 'I)
L-Glutamate.(I1)2-oxoslutarate.(lid (R)-2-hydroxyg|utarate,
(IV) (R)-2-hydroxyglutaryI-CoA,(V) glutaconyI-CoA,(VI)
crotop~'l-CoA.
the hydroxyl group is added to the polarised
double bond of the enoyl-CoA at the electron
richer C 2 rather than at C 3. This orientation is
just opposite to that in the well-known hydration
of crotonyl-CoA to (S)-3-hydroxybutyryl-CoA
catalysed by crotonase [10].
In the following section of this article, these
(R)-2-hydroxacyl-CoA dehydratases are reviewed.
Thc two consecutive sections deal with 4-hydroxybutyryl-CoA dehydratase and 5-bydrox,yvalerylCoA dehydratase which have to cope with similar
mechanistic problems. The final section reports
on a novel serine dehydratase containing iron and
sulfur.
There are two additional unusual dehydratases
acting on 1,2-diols, such as propanediol, ethyl-
DEHYDRA-
Three different 2-bydroxyacyi-CoA debydratases have been purified in recent years. The
enzyme from C. propionicum catalyses the dehydration of (R)-lactyl-CoA, derived from alanine
(Fig. 2) or serine, to acryloyl-CoA [8,14] as well as
(R)-2-hydroxybutyryl-CoA (from threonine) to
crotonyl-CoA [15]. (R)-2-Hydroxyglutaryi-CoA
(from glutamate, Fig. 3) is dehydrated to (E)glutaconyl-CoA by enzymes from A. fermentans
[7] and Fusobacterium nucleatum (A.-G. Klees
and W. Buckel, unpublished). The dehydration of
(R)-phenyllactyl-CoA (from phenylalanine) to
cinnamoyI-CoA (Fig. 4) has been studied in ceilfree extracts of C. sporogenes [16]. However, only
indirect evidence is available for the dehydration
of (R)-4-bydroxyphenyilactyl-CoA(from tyrosine),
(R)-indollactyl-CoA (from tryptophan) and (R)2-hydcoxy-4-metbylvaleryi-CoA (from leucine) in
C. sporogenes and a few other clostridia [17-19].
3.1. (R)-Lactyl-CoA dehydratase
In 1947 Cardon and Barker reported the fermentation of alanine, lactate and acrylate to propionate and acetate by C. propionicum [20]. Both
fatty acids were also formed from lactate and
acrylate by Megasphera elsdenii, formerly called
rumen coccus LC [21]. From these observations
as well as from '4C-tracer studies it was concluded that lactyl-CoA is dehydrated to acryloylCoA in both organisms [22] (Fig. 2). This pathway
was called non-randomising, since the carbon
skeleton of lactate remained unchanged in propionate. In contrast, the fermentation of lactate to
propionate by propionibacteria involves the sym-
214
l
1
H
H
I NADH
z..--~ II ~.,,. !1
I 2W
H
CO0.
S ,, n* co~coA~
CO0"
~
CO0"
cosco~t
N
Phenyl*ltnlne ÷ 2 H
• Phenylproplola*te"+ NH4*
Fig. 4. Reductionof phenylalanineby Clostridiumsporogenes.
(I) Phenylalanine,(Ii) phenylpyruvate,(lid (R)-phenyllactate,
(IV) (R)-phenyllac~l-CoA,(V) einnamoyl-CoA,(VI) cinna
mate, (VII) phenylpropionate.
metrical intermediate succinate leading to randomisation of C2 and C3 [23].
Early attempts failed to demonstrate the dehydration of iactyl-CoA to acryloyl-CoA in cell-free
extracts prepared from C. propionicum [24]. Only
extracts from pigeon heart muscle and from
Pseudomonas sp. grown aerobically on propionate
were able to hydrate acryloyl-pantetheine to
laetyl-pantetheine [25]. Unfortunately, these interesting aerobic hydrations were not characterised further.
On the other hand, Ladd and Walker [26]
showed that dialysed ceil-free extracts of M. elsdenii catalysed the fermentation of lactate or
acrylate to propionate, acetate and molecular hydrogen in the presence of CoASH and 'sparkling'
amounts of acetyl phosphate under strict anaerobic conditions. The fermentation was inhibited by
low concentrations of hydroxylamine (0.1 raM),
sodium azide (2 mM) and 2,4-dinitrophenol (20
#M). A possible function of 2,4-dinitrophenol as
uncoupler was excluded since an apparent mem-
brane-free 144000 × g supernatant of the cellfree extract had the same catalytic efficiency as a
14000 × g supernatant.
An apparent partial purification of a lactylCoA dehydratase (EC 4.2.1.54) from M. elsdenii
was reported in 1965 [27]. Due to the complicated, coupled assay it is not clear, however,
whether the hydration of acryloyl-CoA to lactyiCoA was actually measured. Furthermore, the
activity was observed under aerobic conditions
under which lactyl-CoA dehydratase from C. propionicum [8,14] was immediately inactivated.
Moreover, a report on the transient formation of
phospholactyl-CoA was not substantiated [28].
The first direct demonstration of the dehydration of lactyl-CoA was the formation of 3HOH
from (R)-[3-3H]lactate in the presence of CoASH
and acetylphosphate catalysed by cell-free extracts of (7. propionicum under strict anaerobic
conditions [8]. Furthermore, the product acrylate,
derived from acryloyl-CoA due to the action of
propionate CoA-transferase [8], was isolated as
;-bromophenacyl ester and identified by mass
spectroscopy [29]. Interestingly, the same compounds used by Ladd and Walker in cell-free
extracts of 114. elsdenii [26] (see above) as were
hydrozylamine (1 raM), azide (1 raM) and 2,4-dinitrophenol (0.1 mM) also inhibited the formation of 3HOH from (R)-[3-3H]lactate. Since the
assay was performed in the pi,~,,ace of ~,6 mM
acetyl phosphate, its removal by 1 mM hydroxylamine can be excluded as a possible reason for
the inhibition.
An enzyme system able to catalyse the hydration of acrylyl-CoA to (R)-lactyl-CoA was purified to homogeneity from cell-free extracts of C.
propionicum by Kuchta and Abeles in 1985 [14].
The system consists of two proteins, component
E I and E II, both of which are sensitive towards
oxygen, especially component I with a halt:life of
< 60 s under air. The successful purification was
only possible by the use of an anaerobic chamber
which is an absolute requirememt for handling all
2-hydroxyacyl-CoA dehydratases. Component E I
is apparently a single polypeptide with a molecular mass of 27 kDa. No prosthetic groups have
been detected on this protein. Hence, the reason
for its oxygen sensitivity remains obscure. Corn-
215
ponent E 11 is composed of two different subunits
(a, 48 kDa; /3, 41 kDa) in a 1:1 ratio. The
molecular mass of the native enzyme was estimated to be about 106 kDa after separation on a
Sepharose 6B column. In the author's laboratory,
however, a value of 260 kDa was determined
using Superose 6 [15]. Thus the quaternary structure of component E I I is most likely a hexamer
a3/33. The enzyme contains "0.5 mol FMN and
0.5 tool riboflavin per tool E I I " [14], most probably per dimer. According to the absorption spectrum of E I I , both flavins are in the reduced
state. Finally, about 8 mol Fe and 8 tool inorganic
sulfur per tool of E I I were detected. EPR measurements indicated the presence of two diffe,ent
iron-sulfur clusters, an unusual [4Fe-4S] and a
[3Fe-4S] cluster. The signal of the latter was
dramatically changed upon addition of either
lactyl-CoA or acryloyl-CoA [30].
The hydration of acryloyl-CoA to (R)-lactylCoA required the presence of both components,
E I and E I I , high levels of Mg 2+ (7 mM) and low
concentrations of ATP (03 raM) [11]. The latter
was already known to be necessary for the activation of (R)-2-bydroxyglutaryl-CoA dehydratase
[31] (see section 3.2). In a typical experiment, 0.4
mM lactyl-CoA was formed from 0.7 mM acryloyi-CoA, indicating the catalytic nature of ATP.
The triphosphate could be replaced by GTP but
not by ADP or AdoPP[NH]P, a non-hydrolysable
analogue of ATP [11]. Thus, ATP hydrolysis is
apparently involved in the activation.
Work in the author's laboratory essentially
confirmed the data on lactyl-CoA dehydratase
[15]. A different assay, however, demonstrated
the reversibility of the reaction. An incubation of
pure component E II, partially pure component
E I, MgCl 2, acetyI-CoA ap.d pure propionate
CoA-transferase catalysed the release of all three
methyl hydrogens from [3-3H]lactate into the
medium. Therefore the reaction must have proceeded forward and backward until all the tritium
had been eliminated. The failure to detect acryloyl-CoA formed from lactyl-CoA [14] is probably
due to the unfavourable equilibrium. It was calculated that K = [lactyl-CoA]/[acryloyl-CoA]= 363
(S.L. Miller, personal communication). This high
value agrees well with the only 0.5% conversion
of (R)-lactate to acrylate catalysed by cell-free
extracts of C. propionicum [29].
(R)-2-Hydroxybutyrate was converted to crotonyl-CoA under the conditions employed to release 3HOH from [3-3H]lactate. CrotonyI-CoA
was identified by oxidation to two moi acetyl
phosphate in a coupled assay using an enzyme
system from A. fermentans [32] (Fig. 3). The
dehydration of (R)-2-bydro~butyryl-CoA to
crotonyl-CoA explains the failure to incorporate
[m4C]acetate into the final product butyrate during the fermentation of threonine by growing
cells of C. propionicum [33]. The carbon skeleton
of threonine remains unchanged in butyrate as
opposed to its synthesis from two mol acetyl-CoA.
The use of (R)-2-hydro~butyryl-CoA as substrate for lactyl-CoA debydratase facilitated the
elucidation of the stereochemicai course of the
reaction. Two stereochemically different samples
of (2R)-2-hydroxy[3-3H]butyrate were prepared
from L-[3-3H]threonine using either threonine
dehydratase from C. propionicum or L-serine dehydratase from Peptostreptococcus asaccharolyticus each in combination with o-lactate dehydrogenase and NADH (see section 6). The result was
clearcut, in both samples only the 3Si-bydrogens
were eliminated. Since the product crotonyl-CoA
has E-configuration, the dehydration occurs in a
syn-mode [15].
3.2. (R)-2-Hydroxyglutaryl-CoA dehydratases
The classical pathway of glutamate fermentation to acetate and butyrate in C. tetanomorphum was elucidated by H.A. Barker [34]. The
pathway involves the coenzyme B~2-dependent
rearrangement of the L-amino acid to (2S, 3S)-3methylaspartate followed by deamination to
mesaconate, hydration to (S)-citramalate and
cleavage to acetate and pyruvate. Finally butyrate
is synthesiscd fro~l 2 mol of the oxoacid. Fermentation of glutamate in P. asaccharolyticus, however, yielded butyrate with the same linear carbon skeleton as the amino acid [35]. Furthermore,
the C 5 dicarboxylates (R)-2-hydroxyglutarate and
(E)-glutaconate rather than the C4 dicarboxylates were detected as intermediates [36,37].
Whereas the pathway via methylaspartate has
216
been found only in a closely related group of
clostridia, the pathway via hydrozyglutarate is
used by a greater variety of bacteria [38,39], the
Gram-positive bacteria P. asaccharolyticus, C.
symbiosum and C. sporosphaeroides as well as the
Gram-negative organisms A. fermentans belonging to the Sporomusa group ([40]; B. Both, personal communication) and Fusobacterium nucleaturn belonging to the Bacteroides group [41].
In a first attempt to elucidate the mechanism
of the dehydration of (R)-2-hydrozyglutarate to
(E)-glutaconate the stereochemical path was determined. Incubation of 2-oxo[3-3H]glutarate in
unlabelled water as well as unlabelled 2-oxoglutarate in 3HOH both in the presence of isocitrate debydrogenase [42,43] followed by glutamate dehydrogenase and NADH afforded
(2S,3R)- and (2S,3S)-[3-3H]glutamates. Fermentation of these stereospecific labelled species with
whole cells of A. fermentans or C. symbiosum
resulted in butyrates clearly showing the release
of the 3Si hydrogen. Since only the E-isomer of
~,lutaconate but not its Z-isomer was fermented
by whole cells of A. fermentans it was concluded
that the dehydration of (R)-2-bydroxyglutarateto
(E)-glutaconate occurred in a syn-mode. Experiments with stereospecifically labelled (2R)-2-hydrozy[3-3H]glutarates completely corroborated
the results obtained with the glutamates [44].
Dialysed cell-free extracts from A. fermentans
catalysed the reversible dehydration of (R)-2-hydrozyglutarate to (E)-glutaconate (but not to the
Z-isomer) under strict anaerobic conditions in the
presence of acetyl phosphate, CoASH, MgCi 2
and NADH [44]. The hydration of glutaconate
was assayed from samples in which (R)-2-hydroxyglutarate was determined enzymatically using crystalline 2-hydrozyglutarate debydrogenase
[44,45]. A more rapid assay determined the 3HOH
released from (2R)-2-hydrozy[3-3H]glutarate. It
was found that MgEDTA, a,a'-dipyridyl or ophenantroline were inhibitory. The activity could
be specifically restored by ferrous ion. Remarkably, the inhibitors for the interconversion of
lactate and acrYlate discovered by Ladd and
Walker [26] (see section 3.1) were also effective in
this system. In additon, 10/zM carbonylcyanide
p-trifiuoromethoxyphenyihydrazone (FCCP) was
the most potent inhibitor [44]. Like 2,4-dinitrophenol, FCCP did not act as uncoupler since
a 200000×g supernatant retained the activity
[31]. The location of (R)-2-hydroxyglutaryl-CoA
dehydratase in the cytoplasm was confirmed later
using the antibody-gold technique [46]. The activity of the debydratase was stimulated, however,
by boiled membranes due to the RNA content. It
was then found that AMP, ADP or ATP were
much more effective than RNA. Other nucleotides were not as stimulatory as those containing adenosine. AMP was probably released
from RNA by er, dogenous RNases present in the
crude system. When ATP was used in the presence of acetyl-CoA, no acetyi phosphate was required anymore for debydratase activity. Hence,
ATP is a new cofactor of the dehydratase [31]. In
contrast, NADH is not a specific reductant for
the enzyme. Later NADH was replaced by ferrous ion and dithiothreitol or, more efficiently, by
Ti(IIl)citrate.
For initial attempts to purify (R)-2-hydrozyglutaryl-CoA dehydratase cells from the late logarithmic growth phase of A. fermentans were used.
At this stage the enzyme showed activity without
added ATP. It could be localised by its activity in
the effluent of a Mono Q column, though in low
yield. A better yield was obtained by chromatography of an extract derived from early stationary
cells on Q-Sepharose. Activity was only observed
by adding ATP and the flow-through of the column. Therefore the latter fraction was designated
as 'activator', whereas the main component retarded by the column was named (R)-2-hydroxyglutaryi-CoA dehydratase. The final purification
of the dehydratase was achieved by chromatography on Blue-Sepharose. Although the enzyme
was pure as judged by SDS-PAGE, the specific
activity was low (0.93 nkat mg "l) and only five
times higher than that of the crude extract, indicating the purification of mainly inactive protein.
The dehydratase is composed of two different
subunits (a, 55 kDa; g 42, kDa) arranged in a
heterotetramer (a2/32, 210 kDa). The enzyme
contains 8 mol iron and 8 tool inorganic sulfur
per tetramer [7]. Later reduced riboflavin was
also detected in a debydratase preparation which
was purified by Mono Q instead of Blue-Sep-
217
harose [47]. Obviously, the dye of the affinity
column had replaced the riboflavin.
The activator was purified by affinity chromatography on ATP-agarose but the preparation
contained several proteins as analysed by SDSPAGE. Thus, only the apparent molecular mass
of the native activator could be determined as 56
kDa. In the activation reaction, ATP could not be
replaced by its non-hydrolysable analogues
AdoPP[NH]P, AdoPP[CHe]P or AdoP[CH2]
PP. The former also inhibited the activation by
A T E The activator preparation rapidly hydrolysed ATP to ADP followed by a slower formation
of adenosine. By incubation of [a-32P]ATP or
[~-32P]ATP witil activator and dehydratase no
radioactivity was incorporated into the protein
[7,471.
Purified (R)-2-hydrox'yglutaryI-CoA dehydratase was not able to catalyse the dehydration
of (R)-2-hydroxyglutarate even in the presence of
acetyl-CoA, MgCI2, dithiothreitol, ferrous ion,
activator and ATP. The system required a third
enzyme, glutaconate CoA-transferase, which was
purified from A. fermentans and crystallized [48].
The enzyme catalyses the transfer of CoAS from
acetyl-CoA to (E)-glutaconate > glutarate > (R)2-hydroxyglutarate :~ (S)-2-hydroxyglutarate = 3hydroxyglutarate (increasing apparent Kin); no
activity was observed with (Z)-glutaconate. The
requirement of glutaconyI-CoA transferase indicated for the first time that (R)-2-hydroxyglutaryl-CoA was dehydrated to (E)-glutaconylCoA on the thiolester level rather than as a free
acid [7]. Hence, the questiop, on the structure of
(R)-2-hydroxyglutaryl-CoA arose. There are two
isomers, (R)-2-hydroxyglutaryl-l-CoA and -5CoA, which are generated by glutaconyl-CoA
transferase [48] and are separable by HPLC [49].
Independent chemical ':ynthesis of (R)-2-hydroxyglutaryl-l-CoA demonstrated that only this isomer was accepted by the dehydratase. It was also
kinetically favoured by glutaconate CoA-transferase whereas the 5-isomer was the thermodynamically favoured product due to the higher
pK a of the corresponding acid [49].
A genomic library of A. fermenmns DNA constructed with the lambda vetor EMBL 3 was
amplified in Escherichia coli and screened with
an antiserum against (R)-2-hydroxyglutaryI-CoA
dehydratase. A clone containing the genes for
both subunits, hgdA (a-subunit) and hgdB ([3subunit), was thus obtained and sequenced. The
genes had the following order: A-B, with an intergenic region of only 2 bp. The molecular masses
of the subunits (a, 53870; /3, 41857) calculated
from the deduced protein sequences agreed well
with those determined by SDS-PAGE (55 and 42
kDa, see above). Both subnnits are extremely rich
in cysteine residues (13 in a, including a CNC
and two CC clusters, and nine in/3). No similarities to other known protein sequences were found
[5Ol.
An antiserum prepared against both subunits
of (R)-2-hydroxyglutaryl-CoA dehydratase from
A. fermentans [46] did not i~act in Western blots
with cell-free extracts of the other bacteria able
to ferment glutamate via (R)-2-hydroxyglutarate.
This result was unexpected, since an antiserum
against glutaconyl-CoA decarboxylase, the next
enzyme in the pathway (Fig. 3), detected a relationship between the decarboxylases of these organisms [51]. It was therefore of interest to see
whether the (R)-2-hydroxyglutaryl-CoA dehydratases also differ in other properties. F. nudeaturn was chosen as an enzyme source because of
its large evolutionary distance from A. fermentans. The extremely oxygen-sensitive enzyme was
purified to homogeneity by Q~Scpharose followed
by Superdex and Blue-Sepharose. No activator
was necessary for activity, though ATP stimulated
up to 3-fold. Remarkably, the pure enzyme consisted of three rather than two subunits with
molecular masses of 49, 39 and 24 kDa. Possibly,
the smallest subunit represented the activator.
Like the dehydratase from A. fermentans, the
fusobacteriai enzyme contained riboflavin (about
0.5 m o l / l l 2 kDa), iron and sulfur (about 4
mol/|12 kDa each). The activity of the new dehydratase was measured in a continuous assay
through the formation of NADH using purified
glutacony|-CoA decarboxylase and enz~les from
th~ oxidative branch of the pathway (Fig. 3) [321.
Thereby specific activities of up to 500 nkat/mg
protein were determined. The enzyme was inhibited or inactivated by 0.1 mM hydroxylamine, l0
/zM 2,4-dinitrophenol and, interestingly, by 10
218
ItM chloramphenicol (A.-G. Klees and W. Buckel,
unpublished).
3.3. Dehydration of (R).phenyUactyl.CoA to cinnamoyl.CoA
C. sporogenes, C. botulinum and C. caloritolerans are able to reduce phenylalanine to phenylpropionate whereas C. mangenoti, C. ghoni, C.
bifermentans and C. sordellii form additional
phenyllactate from the aminoacid [17,52]. Later it
was shown that resting cells of C. sporogenes are
able to reduce L-phenylalanine, phenylpyruvate,
(R)-phenyllactate and (E)-cinnamate to phenyipropionate under an atmosphere of molecular
hydrogen. From these results it was concluded
that in the course of the fermentation of phenylalanine, the phenyllactate (2-hydroxy-3-phenylpropionate) was dehydrated to cinnamate [18].
Thus the reductive branch of the pathway of
phenylalanine fermentation was established (Fig.
4) [19]. The pathway is very similar to that of the
conversion of alanine to propionate (Fig. 2), the
exception being the reduction of free cinnamate
rather than cinnamoyi-CoA to phenylpropionate
catalysed by enoate reductase [53].
The stereochemical course of the elimination
of water from (R)-phenyllactate was analysed with
(2R,3R)-phenyl[3-3H]lactate and (2R,3S)phenyl[3-3H]lactate synthesised from phenylpyruvate using phenylpyruvate tautomerase and an
NADH-dependent 2-oxoacid reductase from C.
sporogenes. Incubation of the labelled substrates
with whole cells of the same organism yielded
phenylpropionates, showing that the 3Si-bydrogen had been removed during the elimination of
water. Assuming that the E-configuration of cinnamate was formed directly from (2R)-phenyllactate, the dehydration occurred in a syn fashion
[54]. Hence all analysed (R)-2-bydroxyacyl-CoA
dehydratases operate by the same geometry.
Experiments in ceil-free extracts of C. sporogenes demonstrated the requirement of acetylCoA or ATP as well as acetyl phosphate in combination with CoASH. Thus (2R)-phenyilactylCoA rather than the free acid was the substrate
for the dehydration. Activation of the enzyme by
ATP was not shown. Remarkably, 1 mM hydroxylamine inhibited the dehydration [16].
3.4. On the mechanism of action of 2-hydroxyacylCoA dehydratases
The mechanism of action of 2-hydroxyacyl-CoA
dehydratases is an intriguing question. There are
chemical models for inversions of the orientation
in the addition of HX to asymmetric double bonds
as found with the hydration of acryloyl-CoA and
glutaconyl-CoA. An example illustrating such an
inversion is the addition of HBr to the asymmetric double bond of propylene. In the absence of a
catalyst the proton adds to the carbon that has
more hydrogens, yielding /so-propyibromide following the rule of Markovnikov. The reason is the
more stable carbocation at C 2 ~'ather than at C i.
Under light or in the presence of peroxides, however, bromo radicals are formed which attack at
C~ for stericai reasons and n-propylbromide, the
anti-Markovnikov product, is formed [55]. It is
noted that the use of Markovnikov's rule is confusing in the case of the highly polarised acryloyiCoA. The hydration of acryloyl-CoA to 3-hydroxypropionyl-CoA rather than to lactyl-CoA
would be a violation of the rule. Furthermore, in
the case of glutaconyl-CoA the rule cannot be
applied at all. Nevertheless, examples like the
hydrobromation of propylene, prompted to propose the involvement of radicals in the dehydrations of 2-hydroxyacyl-CoA derivatives. Especially, in the first step a radical abstraction of the
non-activated hydrogen at C 3 was suggested
[7,14,31]. An attractive hypothesis was the formation of a 5'-deoxyadenosine radical from ATP as
reactive species [56,57]. Such a species has been
shown to be generated from coenzyme B n [1113,58] or from S-adenosylmethionine [59] during
enzymatic catalysis. In both cases, 3H from the
5'-position of the adenosine moiety was transferred into the product. However, no 3HOH was
formed from [2,8,5'-3H]ATP during dehydration
of (R)-2-11ydroxyglutaryi-CoA [47]. In addition, a
migration of the hydroxyl group from C 2 to C 3
initiated by a 5'-deoxyadenosine radical like in
the coenzyme Bt2-dependent diol debydratase [11]
would yield 3-hydroxyacyi-CoA, the dehydration
of which is well understood [4,5]. However, /3lactate (3-bydroxypropionate) and 3-hydroxyglutarate were not fermented by cell-free extracts
of C. propionicum [14] and ,4. fermentans [44],
219
respectively, whereas (R)-iactate and 2-hydroxyglutarate were fermented readily. The 3-hydroxy
acids were certainly activated to the CoA esters
under these conditions since the corresponding
CoA-transferases do not exhibit high specificities
[14,48,60]. Thus 3-hydroxyacyl-CoA esters are no
substrates for the corresponding 2-hydroxyacylCoA dehydratases.
No evidence for the involvement of radicals in
the dehydration of (R)-lactyl-CoA or (R)-2-hydroxyglutaryl-CoA has been obtained by EPRspectroscopy ([30]; A-G. Klees and W. Buckel,
unpublished). The free radical observed in partially purified (R)-2-hydroxyglutaryi-CoA dehydratase [47] was shown to be due to contamination by a flavin radical (R. Dutscho and W.
Buekel, unpublished). From stereochemical investigations there is also no evidence for involvement of a radical centered at C 3 of (R)-2-hydrox~butyryl-CoA. The confirmation of a radical
intermediate would be expected to be not completely controlled by the enzyme [61], leading to
racemisation. However, racemi~ation'was not observed.
The failure to detect free l adicals and the
absence of coenzyme B t 2 a s well as the presence
of [Fe-S] clusters and reduced riboflavin in (R)2-hydroxyacyl-CoA dehydratases may lead to a
totally different mechanism (Fig. 5a). The overall
H
V ..~,,oa
CoAS~
C~
C
q
o
Y
H
I
V ~u
CoAS~C~C~
~OH-
Y
CoAS - -
I
C--
C
"
H
z
CoAS - -
H
H
\o /
Y
H-o ........
H
I
H
I
I
C --
C-'--C,~"
H
'
O"
!I~
0
Fig. 5. A. Hypothetical mechanism for the dehydration of
(R)-2-hydroxyacyl-CoA with saturated acyl-CoA as intermedi-
ate (first version). B. Hypothetical mechanismlot the dehydration of (R)-2-hydroxyacyI-CoAwith an epoxideas intermediate (secondversion).
Co~ ~ C ~ C ~ c ~ "
It
o
Fig. 5. (continued).
/
H
H
220
syn-elimination observed in all examples could be
a two-step redox reaction, a reduction followed
by an oxidation. Thus a nucleophilic attack at C2
by a hydride from the reduced riboflavin would,
in an SN2-displacement, yield the saturated thiolester and a hydroxyl ion which could be bound to
an iron atom of the [Fe-S] clluster like in aconitase [62]. A chemical model for this displacement
could be the reduction of mesitylated ethyl (S)lactate by LiAIeH4 yielding (S)-[1-2He,2-2H]propanol with inversion of configuration [63]. In the
second step the now oxidized riboflavin would
dehydrogenate the saturated thiolester to the final product enoyl-CoA whereby the reduced riboflavin would be regenerated. The mechanism
explains the necessity of the activation of the
acids to the CoA-esters in two ways. Firstly, the
reactivity of the hydroxylgroup is enhanced by the
adjacent thiolester. Secondly, the oxidation is also
facilitated at the CoA-ester level. The proposed
mechanism is consistent with the observed overall
syn-elimination. An inversion of configuration in
the first step should be followed by an antielimination of a pair of hydrogens from C 2 and
C3. AcyI-CoA dehydrogenases as well as enoate
reductases catalyse anti-eliminations [64].
There are two weak points, however, in the
non-radical mechanism proposed above. Firstly,
the oxidation of the saturated CoA-derivatives,
propionyl-CoA and glutaryl-CoA, could not be
demonstrated with the corresponding dehydratases in the presence of artificial electron acceptors (A.E.M. Hofmeister, A.-G. Klees and W.
Buekel, unpublished). Secondly, in the pathway
of alanine fermentation (Fig. 2) it would be pointless to oxidize the proposed intermediate propi-
~00 °
onyl-CoA to acryloyI-CoA which would again be
reduced to propionyl-CoA in the next step. These
considerations lead to another alternative mechanism also requiring thiolesters and reduced flavin
(Fig. 5b) (Sir John Cornforth, personal communication). The reduced riboflavin may act as a nucleophile and add to the thiolester carbonyl of
acryloyl-CoA in a process known as C4a-addition
[65]. In the resulting adduct the reactivity of the
double bond has been reversed or "umgepolt"
[66]. The oxoanion is able to stabilise a carbocation at C 2 produced by the addition of a proton
at C3. Consequently, an epoxide is formed which
then is hydrolysed by water in an SN2-displacement yielding the final product lactyl-CoA. There
is excellent chemical analogy for this process: the
reaction of a-haloketones with alkoxides to form
epoxy-etbers that are solvolysed by carboxylie
acids to a-acyloxyketones [67].
Speculations on the mechanism of the dehydration of 2-hydroxyacyl-CoA esters must take
account of an analogous reaction in which phosphate rather than water is eliminated: the anti-l,4
elimination of phosphate from 3-enolpyruvoylshikimate 5-phosphate to yield chorismate which
is mechanistically equivalent to a syn-l,2 elimination (Fig. 6) [68,69]. Chorismate synthases
catalysing the dephosphorylation were isolated
from plants, moulds and bacteria. Interestingly,
the enzymes from all sources require or contain
reduced flavin, preferentially FMNH-. In the case
of the Neurospora crassa and Bacillus subtilis
enzymes the reduction of the flavin is performed
by an intrinsic diaphorase activity using NADPH
[70]. The aroC genes encoding chorismate synthase from Salmonella typhi and Escherichia coli
~0
o
+ s*PO'.
" ~ ' , -~
0
COO-
OH
.
coo-
=
OH
~-Pheepho-~enolpyruvoyl-shlkimate
Chorismate
Fig. 6. Reactioncatalysedby chorismatesynthase.
have been cloned and sequenced [70,71] but no
homology to the hgd genes encoding (R)-2-hydroxyglutaryl-CoA dehydratase from A. fermentans has been detected. Contrary to the 2-bydroxyacyl-CoA debydratases, the chorismate synthases do not require activation by ATP nor a
CoA-derivative as substrate. Therefore, one is
tempted to assume a phosphorylation of the hydroxyl groups of lactyi-CoA and 2-hydroxyglut~ryl-CoA by ATP in order to obtain a better
leaving group as suggested by Anderson and
Wot~ [9]. A mechanism is lacking, however, by
which ATP would be regenerated by the dehydratases. On the other hand, the formation of an
epoxide from 3-enolpyruvoyi-shikimate 5-phosphate or chorismate as suggested above appears
to be very unlikely, due to the absence of a
thiolester.
4. 4-HYDROXYBUTYRYL-CoA DEHYDRATASE
-OOC~NH3
+
.~~o
~÷
~
t
w 0 ~
~
0
H
v
A.n,
4-Aminobutyrate is a common non-proteinogenic amino acid formed by decarboxylation of
glutamate. In vertebrate brains, 3,-aminobutyric
acid (GABA) serves as an inhibitory neurotransmitter [72]. 4-Aminobutyrate is fermented to ammonia, acetate and butyrate by the strict anaerobe C. aminobutyricum isolated by Hardman and
Stadtman [73]. Further work of the authors
showed the initial exchange of the amino group
of 4-aminobutyrate by a hydroxyl group through
the combined action of the enzymes 4-aminobutyrate aminotransferase, glutamate dehydrogenase and 4-hydroxybutyrate debydrogenase, all
detected in cell-free extracts of the clostridium
[74,75] (Fig. 7). Subsequently, it was proposed
that the resulting 4-hydroxybutyrate was activated
to the CoA-ester, followed by dehydration to
vinylacetyl-CoA and isomerisation to crotonylCoA. Disproportionation of the latter CoA-derivative would yield the final products acetate and
butyrate [75].
The dehydration of 4-hydroxybutyryl-CoA to
vinylacetyi-CoAposes a mechanistic problem similar to that of the 2-bydroxyacyl-CoA dehydratases. Again a hydrogen has to be removed
Sum: 2 • 4-Amin~l,t,~Tr~te 4- 2 H20 ~
2 NH4 + + Bu~rale-+ 2 Acetate-+ H +
Fig. 7. Fermentation of 4-aminobutyrate by Clostridium
aminobutyricum,modifiedfrom[54].(I) 4-Aminobutyrate,(!I)
succinate semialdehyde,(III) 4-hydroxybutyrate,(IV) 4-bydroxybutywl-CoA, (V) vinylace~l-CoA,(VI) crotonyi-C_.oA.
Alternatively,ATP could be formed from acetyiphosphate.
The exactpathwayhas not yet beenestablished.
from C 3 which is not activated by the thiolester.
Consequently, the necessity of the formation of
the CoA-ester was not immediately apparent. On
the other hand, a coenzyme Bn-dependent migration of the hydroxyl group from C 4 to C 3
occurring without hydrogen exchange with the
solvent [11] would yield 3-hydroxybutyryl-CoA
from which a proton at C2 rather than at C3
should be removed. Both alternatives were disproven by the application of 4-bydroxy[3-3H]
butyrate as substrate obtained by heating succinic
semialdebyde in tritiated water followed by reduction with sodium borobydride. Cell-flee extracts of C. aminobutyricum cataiysed the stereospecific release of 49 + 2% of the 3-3H-label into
the medium only in the presence of acetyl-CoA,
suggesting the direct dehydration of 4-bydroxy-
222
[3-3H]butyryi-CoA to [3-3H]vinylacetyi-CoA and
3HOH (Fig. 7) [76].
The CoA-transferase necessary to activate 4hydroxybutyrate by acetyl-CoA was purified 31fold to homogeneity from extracts of C. aminobutyr/cum. The enzyme was relatively specific for
4-hydroxybutyryl-CoA ( k c a t / K m ~ 2.4 s- I /tM- t
in the presence of 200 mM acetate) as compared
to butyryi-CoA (0.50), propionyl-CoA (0.50),
vinylacetyi-CoA (0.26) and 5-hydroxyvaleryl-CoA
(0.06). Crotonyi-CoA as well as DL-3- and DL-2hydroxybutyrate were no substrates. The enzyme
was very useful to prepare 4-hydroxybutyryl-CoA
from acetYl-CoA and 4-hydroxybutyrate, since
chemical methods failed most probably due to
the facile formation of butyrolactone [77].
HH H H
o H H
H
CoAS~
H
H
H
5. 5-HYDROXYVALERYL-CoA DEHYDRATASE
H
CoAS~
H
eo
H
H
CoAS- ~ ~
0
CHs
H
The dehydration of 4-hydroxybutyryi-CoA is
catalysed by a moderately oxygen-sensitive enzyme [76]. It was purified to homogeneity under
anaerobic conditions. The brown protein is a
homotetramer (molecular mass = 230 kDa) containing an [Fe-S] cluster and reduced FAD. The
pure enzyme still showed considerable, probably
intrinsic vinylacetyI-CoA delta-isomerase activity
(U. Scherf and W. Buckel, unpublished). Thus, it
remains to be established whether the only detectable product, crotonyl-CoA, is directly derived from 4-hydroxybutyryl-CoA or via vinylacetyl-CoA. A possible mechanism of the dehydration could involve cyclopropylcarboxyl-CoA
which may be formed in a nucleophilic SN2-displacement of the hydroxylgroup by a carbanion at
the a-carbon (Fig. 8). The 5tereospecific removal
of one of the hydrogens at C3 would then be
facilitated by the electron withdrawing thiolester
whereby the ring would be opened again. Finally,
the formed vinylenol would tautomerise to
crotonyl-CoA or to vinylacetyl-CoA. This mechanism is consistent with the requirement of a
CoA-ester as substrate. Furthermore, the [Fe-S]
cluster could be involved in the elimination of the
hydroxylgroup as in aconitase [62] whereas the
isomerase activity supports the postulated intermediate vinylenol. An open question, however,
remains the presence of FAD in the enzyme.
Using artificial electron acceptors a butyryI-CoA
dehydrogenase activity could not be detected (U.
Scherf and W. Buckel, unpublished).
CoJ~~
H
0
U
Fig. 8. Hypotheticalmechanismof the dehydration of 4-hydroxybutyryI-CoA.
The next homologue of 4-hydroxybutyryl-CoA
is 5-hydroxyvaleryl-CoA which was discovered by
H.A. Barker [78] as an intermediate in the fermentation of 5-aminovalerate to n-valerate, propionate and acetate by the strict anaerobe C.
aminovalericum [79]. The non-proteinogenous
amino acid 5-aminovalerate is generated from
proline in the reductive branch of a Stickland-type
fermentation in C. sporogenes and some other
anaerobes [80,81]. The conversion of 5-aminovalerate to 5-hydroxyvaleryl-CoA exactly follows
223
the same pathway as that of 4-aminobutyrate to
4-hydroxTbutyryl-CoA (Fig. 9). The consecutive
crucial step, the dehydration of 5-bydroxyvalerylCoA to 4-pentenoyl-CoA, was shown to proceed
in cell-free extracts. It was measured in the reverse direction under aerobic conditions [78].
Again the requirement of a CoA-ester substrate
is not immediately apparent since the hydrogen
to be removed from C4 of 5-hydroxyvaleryi-CoA
is separated by two methylene groups from the
~H~N~
thiolester. In this case, however, an extra double
bond can be introduced between C2 and C3
whereby C4 becomes connected to the electron
withdrawing force of the thiolester. Thus, 5-hydroxyva;eryl-CoA should be oxidised to 5-hydroxy-2-pentenoyI-CoA, dehydrated to 2,4-pentadienoyI-CoA and finally reduced to 4-pentenoylCoA (Fig. 9).
T'-,is proposed mechanism was confirmed by
using purified enzymes. Firstly, 5-hydroxyvalerate
COO~ - 2-0zoglutsr.te~(,..-P NADH+ r +~-~
l
~x~.~ Glutamate~ "
o~
~
NAI~+ IlaO
coo-
If-NADH + H*
II
t
coo"
HO ~
Acetyl-CoA
III
Pr0pionyI-CoA4,,,,
~CoASH
~ ~ - . - - - ~ [ Propioute÷ Valerate [
COSCoA
W
XII ~:
~
,~o ~ ~ c o s c o S ' ~
v
COSCoA
~
COSCoA
Vl
O
~COSCoA
NAD*,,I
~T
Naps
-I
XI
~
COSCoA
/
lu]
[
COSCoA
Vlll
~
~
COSCoA
IX
COSCoA
VII
AYP
/k
Sum:2 x ~-Amiaov~lerate÷ 2 HsO 7 ~ 2 H, ÷ ÷ Valerate'÷Pr0piouate-÷Acetate-+r
Fig. 9. Fermentation of 5-aminovaleratc by Clostridium aminoealedcum, modified from [54]. (I) 5-Aminovaleratc,(II) glutarsemialdehyde, (lid 5-hydroxyvalerate,(IV) 5-bydroxyvalcryi-CoA,(V) (E)-5-hydroxy-2-pentenoyi-CoA,(Vl) (E)-pentadienoyl-CoA,
(VII) 4-pcntenoyl-CoA,(vm) (E)-3-pentenoyl-CoA,(IX) (E)-2-pentcnoyl-CoA,(X) (S)-3-hydroxyvalcryl-CoA,(Xl) 3-oxovalcrylCoA, (XII) valcryI-CoA.
224
CoA-transferase was obtained as a homogenous
protein [82]. The enzyme is specific for hydroxy
acids or their CoA-derivative~, preferentially 5hydroxyvalerate, as well as for unsubstituted fatty
acids, preferentially propionate. Important for the
elucidation of the mechanism was the reactivity
of the CoA-transferase towards (Z)-5-hydroxy-2pentenoate and with less efficacy towards 4pentenoate.
Secondly, a green 5-hydroxyvaleryi-CoA dehy-.
dratase was purified from ceU-free extracts of C.
aminovalericum and crystallised [83]. The crystals
are suitable for X-ray crystallography to 0.2 nm
resolution (E.F. Pal, U. Eikmanns and W. Buckel,
unpublished). The homotetrameric enzyme contains 4 tool FAD/169 kDa and 4 mol of a CoAester similar but not identical to 5-hydroxy-2pentenoyl-CoA. The unusual ultraviolet/visible
spectrum of the green enzyme (maxima at 394
nm, 438 nm and 715 nm) was converted to a
normal flavoprotein spectrum either by reduction
with dithionite and reoxidation under air, or by
removal of the prosthetic group at pH 2 and
reconstitution with FAD. The reconstituted yellow holoenzyme as well as the native green enzyme, but n0t the apoenzyme, catalysed the reversible dehydration of 5-hydroxyvaleryl-CoA to
4-pentenoyi-CoA in the absence of an external
electron acceptor. In its presence, preferentially
ferricenium ion, the green or yellow enzyme
catalysed the formation of (E)-5-hydroxy-2pentenoyl-CoA and 2,4-pentadienoy!-CoA either
from 5-hydroxyvaleryl-CoA or from 4-pentenoylCoA. The reversible hydration of 2,4-pentadienoyl-CoA to (E)-5-hydroxy-2-pentenoyl-CoA was
mediated by both holoenzyme forms as well as by
the apoenzyme in the absence of FAD. Hydration
of 4-pentenoate in 2H20 yielded optically active
5-hydroxy(2,4-2H2)valerate by the combined action of 5-hydroxyvalerate CoA-transferase, the
green dehydratase and catalytical amounts of
acetyl-CoA. The incorporation of deuterium at
C4 is consistent with the hydration of the double
bond between C4 and Cs whereas the incorporation of deuterium at C 2 indicates the transient
formation of the double bond between C2 and
C 3. A lack of exchange at C 3 was also found with
the FAD-containing general acyl-CoA dehydro-
genase [84]. In summary, the data show that the
reversible anti-Markovnikov hydration of the isolated double bond of 4-pentenoyl-CoA to 5-hydroxyvaleryl-CoA is preceded by the oxidation to
2,4-pentadienoyl-CoA. The latter compound, a
vinyl analogue of 2-enoyl-CoA, is then easily hydrated to (E)-5-hydroxy-2-pentenoyl-CoA and finally reduced to 5-hydrox,yvaleryI-CoA [83] (Fig.
9).
The formation of 4-pentenoyl-CoA from 5-hydroxyvaleryl-CoA catalysed by the dehydratase
proceeded at the low rate of 0.3 s -t, whereas in
the presence of ferricenium ion a mixture of
5-hydroxy-2-pentenoyl-CoA and 2,4-pentadienoyl-CoA was produced at a 40-times higher rate.
Therefore it was concluded that in vivo the product derived from 5-hydroxyvaleryl-CoA was 2,4pentadienoyl-CoA rather than 4-pentenoyl-CoA
[83]. This agrees well with the detection and
purification of a green, FAD-containing enzyme
in C. arrdnovalericum catalysing the reduction of
2,4~pentadienoyl-CoA to 3-pentenoyl-CoA [85]
(Fig. 9). Attempts failed, however, to identify an
electron carrier connecting the dehydratase with
the reductase; NAD + and NADP + were ineffective (C. Mirwaldt and W. Buckel, unpublished).
Cell-free extracts of C. aminovalericum contain a
very active delta-isomerase catalysing the shift of
the double bond of 3-pentenoyl-CoA to 2pentenoyl-CoA [78]. Disproportionation of the
•latter by the ~-oxidation pathway yields valerylCoA as well as propionyl-CoA and acetyl-CoA.
The two longer CoA-esters are used to activate
5-hydroxyvalerate whereas ATP is generated via
acetyl-CoA and acetyl phosphate [78].
6. L-SERINE DEHYDRATASE
L-Serine dehydratases catalyse the overall
deamination of L-serine to pyruvate. L-Serine dehydratases and the related threonine dehydratases are ubiquitous enzymes found in animals
as well as in aerobic and anaerobic bacteria. The
initial step in catalysis is a/3-elimination of water
followed by tautomerisation and hydrolysis to ammonia and pyruvate or 2-oxobutyrate, respectively
225
(Fig. 1; reaction II). It is not immediately apparent that this kind of dehydration is unusual since
the proton to be removed from the a-carbon is
activated by the carboxyl group. Furthermore, all
previously described L-serine dehydratases contain pyridoxal phosphate forming ~ Schiff base
with the amino group of serine and leading to an
even more acidic hydrogen at the a-carbon.
Recently, however, an L-serine dehydratase
devoid of pyridoxal phosphate was discovered in
the strict anaerobe Gram-positive bacterium P.
asaccharolyticus [86]. The enzyme was inactivated
by exposure to air. But the activity could be
restored by incubation with ferrous ion. The active enzyme contained about 4 mol iron and 5-6
mol inorganic sulfur/55 kDa indicating the presence of an iron-sulfur cluster. Preliminary EPRspectroscopy of the active enzyme showed the
signal of a high potential [4Fe-4S] cluster which
was converted to that of a [3Fe-4S] cluster upon
inactivation (A.E.M. Hofmeister, R. Grabowski
and W. Buckel, unpublished). Another difference
to the 'normal' pyridoxal pbosphate-containing
enzymes is the composition of two different subunits (a, 30 kDa; /3, 25 kDa) which are most
likely arranged in a hetero-octamer (200 kDa)
[86]. Finally, the stereochemistry of the dehydration confirms the distinctness of this novel L-serine
dehydratase from the pyridoxai phosphate-dependent enzymes. Whereas in all pyridoxal phosphate-containing dehydratases the hydroxyl group
is replaced by the hydrogen with retention of
configuration, the iron-sulfur-containing enzyme
catalyses the dehydration of L-threonine with inversion and retention in a 2:1 ratio (Fig. 10). It
was concluded that this racemisation is due to a
release of the product already at the symmetrical
enamine stage whereby the proton is derived
from the solvent rather than from a specific group
of the enzyme. The only partial racemisation suggested that the protonation occurred still in the
vicinity of the enzyme [87].
The reversible i~;terconversion of the [4Fe-4S]
cluster into a [3Fe-4S] cluster may be similar to
that found in aconitase [62]. Hence, one of the
irons in the [4Fe-4S] cluster may not be bound
directly to the protein and might be able to
interact with the hydroxyl group of the substrate
H
U
~- ~0COO-
D
D
~
H
0
Retention
COO-
D
H -"
CO0"
Inversion
Fig. !0. Stereochemicalpathways of the dehydrationof Lthreonine in D20.
serine. Therefore, the enzyme would activate the
hydroxyl group rather than the hydrogen at the
a-carbon as in the pyridoxal phosphate-dependent dehydratases, l~t this respect the enzyme
from P. asaccharolyticus catalyses an unusual dehydration of/3-hydroxyamino acids. Most probably, the enzyme is only the first known member of
a whole family of iron-sulfur-dependent L-serine
dehydratases. There is evidence that enzymes
from C. acidi-urici [88], lactic acid bacteria [89],
E. coli [90] and C. propionicum (A.E.M. Hofmeister and W. Buckel, unpublished) also contain
iron-sulfur clusters. Remarkably, the distinct
threonine dehydratase from the latter organism
appears to be dependent on pyridoxal phosphate
[87]. The available data may lead to the generalisation that the dehydratases highly specific for
serine should be iron-sulfur enzymes whereas
those acting either on threonine alone or on both
hydroxyamino acids should contain pyridoxal
phosphate or another electrophilic center.
226
7. CONCLUDING REMARKS
in a syntrophic culture. Curiously, the reversible
anti-dehydration of (R)-10-hydroxydecanoic acid
This review describes the dehydrations of a
series of hydroxy acids ranging from 2-hydroxy to
5-hydroxy acids as summarized in Table 1. In
most cases the hydroxy acids are generated from
the corresponding amino acids by mere exchange
of the amino group by a hydroxyl group. Apparently, in some reactions the elimination of ammonia is more difficult than that of water. The
dehydrations of 3- and 5-hydroxyacids are fairly
well understood whereas those of the 2- and
4-hydroxyacids are more elusive reactions. Although there is no net oxidation or reduction, the
2- and 4-hydroxyacyl-CoA dehydratases contain a
unique set of redox prosthetic groups: iron-sulfur
clusters and flavins (sections 3 and 4). Remarkably, also certain L-serine dehydratases (section
6) and some other 3-hydroxy acid dehydratases
contain iron-sulfur clusters [91] whereas the 5hydroxyvaleryl-CoA dehydratase contains FAD
(section 5). The function of the flavin has been
elucidated only in the latter enzyme. It may be of
interest to study the dehydration of the next
homologue 6-hydroxycapryl-CoA which should be
mechanistically similar to that of 4-hydroxybutyryl-CoA. An organism fermenting the corresponding amino acid 6-aminocaproate could not
be isolated from sew3ge sludge whereas 5aminovalerate-degrading organisms were easily
obtained from this source. Possibly, 6-aminocaproate is initially degraded by/3-oxidation requiring additional electron acceptors such as sulfate, nitrate or a hydrogen-consuming organism
under the formation of the isolated double bond
of oleic acid catalysed by a cell-free extract from
a Pseudomonas was reported. However, no attempts were made to purify the enzyme or to
study the mechanism [92].
The dehydrations of (R)-2-hydroxybutyryl-CoA
and 4-hydroxybutyryl-CoA represent two new
routes to crotonyl-CoA demonstrating the importance of this central intermediate. Altogether
there are eight paths to crotonyl-CoA (Fig. 11):
(I) Crotonate is converted to crotonyl-CoA by
butyryl-CoA: acetoacetate CoA-transferase from
C. subterminale using butyryl-CoA or another
CoA-ester as second substrate [93]. (ll) (R)Lactyl-CoA dehydratase catalyses the formation
of crotonyl-CoA from (R)-2-hydroxybutyryi-CoA
as described in'section 3.1. (Ill) The dehydration
of (S)-3-hydroxybutyryl-CoAto crotonyl-CoA is a
very common reaction catalysed by crotonase or
enoyl-CoA hydratase [10]. (IV) The dehydration
of 4-hydroxybutyryl-CoA is described in section 4.
(V) The dehydrogenation of butyryl-CoA to
crotonyi-CoA is the first oxidative step in the
/3-oxidation of butyrate. Some bacterial butyrylCoA dehydrogenases are green [45,94] like 5-hydroxyvaleryl-CoA dehydratase (section 5) which is
an acyl-CoA dehydrogenase as well [83]. (VI) The
deamination of (S)-3-aminobutyrate to crotonylCoA is a step in the fermentation of L-lysine in C.
subterminale [93,95]. Interestingly, the amino
group at position 3 is removed directly, whereas
the removal of amino groups from positions 2, 4
Table 1
Moleculardata of the.dehydratasesdescribedin this review
Organism
Closrridiumpropionicurn
Acidaminococcusfermenmns
Fusobacterium nucleatum
Peptostreptococcus
asaccharolytic¢~
CIostridiumaminobutyricum
Clost'ndium aminovalericum
Substrate
Molecular S u b u n i t
Prosthetic
groups
mass
composition
(kDa)
Lactyl-CoA/
270
a3~3
[Fe-S],riboflavin,
2-hydro~butyryi-CoA
FMNH
2-Hydro~glutaryl-CoA 210
a2~ 2
[Fe-S],riboflavin
2-Hydro~glutaryI-CoA 110
agy
[Fe-S],riboflavin
L-Serine
4-HydroxybutyuI-CoA
5-Hydrox~aleryl-CoA
2G~
23C
169
a4~4
a4
a4
[Fe-S]
[Fe-S],FAD
FAD
Cofactors
ATP,activator
ATP,activator
ATP
227
rived from alanine can also be fermented via the
coenzyme Bi2-dependent rearrangement of succinyl-CoA to (R)-methylmalonyl-CoA. Hence, the
dehydrations of (R)-2-hydroxyacyl-CoA derivatives are substitutes for interconversions requiring such a complicated coenzyme as adenosylcobalamin (coenzyme B]2). There is also a third
possibility by which glutamate may be fermented.
Decarboxylation of glutamate yields 4-aminobutyrate which is converted to acetate and butyrate by the pathway described in section 4 (Fig.
7). Although there is hardly any evidence that the
whole pathway occurs in a single organism [97], it
again shows that a chemically difficult dehydration like that of 4-hydroxybutyryl-CoA to
crotonyl-CoA is necessary to ferment glutamate
to acetate and butyrate. Elucidation of the mechanisms of those dehydrations may reveal the advantages and disadvantages of the alternative
pathways for the energy metabolism of the cells.
Recently, W~ichtersh.:iuser developed a theory describing a chemoautotrophic origin of life on
pyrite surfaces [98]. Accordingly, enzymes with
iron-sulfur clusters should represent more primi-
and 5 requires conversion to hydroxyl groups (see
above). (VII) The isomerisation of vinylacetylCoA to crotonyl-CoA is catalysed by enzymes
from various sources, e.g. mitochondria [96], C.
aminovalericum [78] or by pure 4-hydroxybutyrylCoA dehydratase (section 4). (VIII) Finally,
crotonyl-CoA is generated by decarboxylation of
giutaconyl-CoA. The decarboxylase, a key enzyme in the hydroxyglutarate pathway (Fig. 3),
was characterised as a biotin-dependent sodium
ion pump [32]. Recently, it was shown that the
oxidative decarboxylation of giutaryl-CoA to
crotonyl-CoA also proceeded via giutaconyl-CoA
(U. Hiirtel and W. Buckel, unpublished).
The dehydration of (R)-2-h~,~,'oxyacyI-CoA
derivatives is the crucial step in the fermentation
of glutamate via (R)-2-hydroxygiutarate (Fig. 3)
and alanine via acryloyl-CoA (Fig. 2). Interestingly, there are alternatives to both pathways
leading to identical products. As pointed out
above (section 3b), the classical pathway of glutamate fermentation involves the coenzyme Bi2-dependent rearrangement of (S)-glutamate to
(2S, 3S)-3-methylaspartate. Similarly, lactate de-
..~;o
ipl
0
OH 0
~SCoA
f~7oA ~
©rotonyl~A
I!l
o
/
~
0
~SCoA
Vll
o
y
Fig. 11. Reactior~sleading to crotonyI-CoA.(I) Crotonate, (II) (R)-2-hydroxybutyryi-CoA,Illl) (S)-3-hydroxybutyryI-CoA,(IV)
4-hydroxybutyryl-CoA,(V) butyryI-CoA,(VI)(S)-3-aminobutyryI-CoA,(VII)vinylacetyI-CoA,(VIII)glutaconyI-CoA.
228
tive forms w h e r e a s c o e n z y m e BI2 o r pyridoxal
p h o s p h a t e may b e m o r e a d v a n c e d m o l e c u l e s o f
life.
ACKNOWLEDGEMENTS
T h e w o r k p e r f o r m e d in t h e a u t h o r ' s laboratory
was s u p p o r t e d by g r a n t s f r o m t h e D e u t s c h e
Forschungsgemeinschaft and the Fonds der
C h e m i s c h e n Industrie. T h e a u t h o r t h a n k s P r o f e s sor Sir J o h n C o r n f o r t h (Lewes, U . K . ) for discussions o n t h e m e c h a n i s t i c p r o p o s a l s p r e s e n t e d in
this p a p e r . D r a w i n g o f t h e figures by Ms. E l k e
Eckel is gratefully a c k n o w l e d g e d .
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