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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 . REFERENCES [1] IntenTational Union of Biochemistry (1984) Enzyme Nomenclature, Academic Press, New York, NY. [2] Englard, S. (1960) Configurational considerations in relation to the mechanisms of the stereospecific enzymatic hydrations of fumarate and c/s-aconitate. J. Biol. Chem. 235, 1510-1516. [3] Porter, DJ.T. and Bright, H.J. (1980) 3-Carbanionic substrate analogues bind very tightly to fumarase anti aspartase. J. Biol. Chem. 255, 4772-4780. [4] Bahnson, B.J. and Anderson, V.E. (1991) Crotonasecatalysed g-elimination is concerted: A double isotope effect study. Biochemistry 30, 5894-5906. [5] Willadsen, P. and Eggerer, H. (1975) Substrate stereochemistry of the enoyI-CoA hydratase reaction. Eur. J. Biochem. 54, 247-252. [6] Hanson, K.R. and Rose, I.A. 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