Glycine reductase mechanism Jan R Andreesen The ability of some anaerobic bacteria to conserve energy via a soluble substrate level phosphorylation system by reducing glycine to acetyl-phosphate has been an intriguing mechanism for about half a century. The genes implicated in this system have been sequenced and form an operon structure with those of the thioredoxin system. The deduced proteins exhibit high degrees of similarity with glycine reductase from other bacteria. Faster progress in understanding the exact mechanisms is hampered, for example, by some unique reactions involving selenoethers and redox active selenocysteines, which do not allow an easy heterologous formation in Escherichia coli. Further major obstacles are the processing of a substrate-specific pro-protein to a new carbonyl/pyruvoyl group in one of the two peptides formed that stabilize the substrate-binding selenoprotein, which contains an additional rather unstable carbonyl group. Addresses Institut für Mikrobiologie der Universität Halle, Kurt-Mothes-Str. 3, 06120 Halle, Germany e-mail: j.andreesen@mikrobiologie.uni-halle.de Current Opinion in Chemical Biology 2004, 8:454–461 This review comes from a themed section on Mechanisms Edited by Hung-Wen Liu and Jànos Rétey Available online 23rd August 2004 1367-5931/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2004.08.002 Abbreviations BR glycine betaine reductase FDH formate dehydrogenase GR glycine reductase ORF open reading frame Sec selenocysteine SR sarcosine reductase Introduction Only a few groups have worked on the topic of glycine reduction, despite the interest in this unique energy conserving mechanism. Previous reviews [1–3] deal mainly with the system of two Gram-positive anaerobic bacteria, Clostridium sticklandii and Eubacterium acidaminophilum, both of which belong to cluster XI of the clostridia (the same cluster as C. litorale and the pathogenic C. difficile) [4]. Purine degrading clostridia and Tissierella creatinophila are within cluster XII and the Current Opinion in Chemical Biology 2004, 8:454–461 two investigated peptostreptococci belong to cluster XIII. Treponema denticola is the first Gram-negative bacterium known to contain a glycine reductase (GR) system [5], pointing to a much wider distribution within the domain of bacteria. Although thermodynamic considerations allow a substrate level phosphorylation by reductive deamination of glycine with hydrogen 0 (DG0 ¼ 77:8 kJ=mol) [1–3], this unique reaction was not included in many relevant textbooks, probably because it is the first example of a substrate level phosphorylation by a reductive deamination. Usually, dehydrogenation or lyase reactions are involved in such an energy conversion. So far, the transformation of a carboxymethyl selenoether to an acetyl thioester and an oxidized selenoprotein represents a unique reaction involving further selenocysteine-containing proteins as well as thiol groups and still unresolved carbonyl groups as additional essential, but unstable partners in the overall reaction. This review should help to stimulate research and discussion on some still hypothetical reaction mechanisms, and is also in honor to TC Stadtman and RH Abeles, two pioneers on this topic. General constituents of GR and related reductase systems The GR system is generally divided into three protein fractions, called protein A, B and C (Figure 1) [6,7]. Sarcosine reductase (SR) and glycine betaine reductase (BR) exhibit high similarities to GR [8–10]. D-Proline reductase is only partly related to protein B of GR and SR, in particular the features consisting of a selenoprotein and a pro-protein [11,12]. It appears that all relevant genes are now known: protein A (17 kDa, selenocysteine (Sec)-containing) is encoded by grdA [13]; protein B by grdB (47 kDa, Sec-containing) [14] and grdE (which encodes a pro-protein of 48 kDa that will be post-translationally processed into two proteins of 25 and 22 kDa) [15,16]; whereas protein C is encoded by grdC and grdD (54 and 40 kDa, respectively) [12,17]. The reductases for sarcosine and betaine from E. acidaminophilum differ just in the substrate-specific protein B components (grdF and grdG for sarcosine, grdH and grdI for glycine betaine, respectively). Whereas glycine and sarcosine form a Schiff-base with a so-far unspecified carbonyl group of GrdB and GrdF, respectively, the C–N bond of betaine is already polarized as a quaternary amine. Consequently, GrdI protein is no pro-protein [9] and lacks the characteristic two cysteines of the cleavage motif for processing [16]. Figure 1 depicts the involvement of these gene products in the carbon flow from glycine, sarcosine and betaine to www.sciencedirect.com Glycine reductase mechanism Andreesen 455 Figure 1 Protein B Glycine GrdB GrdE Protein A Protein C GrdB-Carboxymethylselenoether Pi SH Sarcosine Betaine GrdF GrdG GrdF-Carboxymethylselenoether GrdH GrdI GrdH-Carboxymethylselenoether Acetyl ~ GrdA GrdC GrdD Acetyl~P Se-CH2-COOH S SH GrdA GrdA Se– Trx Se S S SH Trx SH Current Opinion in Chemical Biology Acetyl phosphate formation from glycine, sarcosine and betaine by E. acidaminophilum, indicating the consecutive involvement of the gene products of respective protein Bs, the integrative action of protein A, and of the final energy-conserving protein C. Thioredoxin (Trx) is the reductant of oxidized protein A [31]. acetyl phosphate. According to the derived amino acid sequences, the different substrate-specific protein Bs for glycine, sarcosine and betaine are highly conserved (using GrdB or GrdE of C. sticklandii as reference), showing about 70%, 59% and 43% identity, respectively, and 76–64% for GrdE proteins from organisms of cluster XI [12]. About the same high identity values are obtained for the universally involved proteins GrdA, GrdC and GrdD. The following strictly conserved motifs are important for the reactions involved: the -UxxCxxC-motif of GrdB, GrdF and GrdH within the -VILTSTUGTCTRCGATMVKEIER/K- sequence [15]; the –CxxU-motif of GrdA is embedded in a -TECFVUTA/S- sequence [14]; the GrdC proteins contain four conserved cysteines at position 173, 223, 228 and 261; and the GrdD proteins include a cysteine at 359 and partially one at 98 (positions according to E. acidaminophilum) [17]. In most organisms, the genes involved in GR form an operon structure that includes genes of the thioredoxin system. The gene grdA is most often located between grdE and grdB for unknown reason, and can be present (as grdB, grdC and grdD) in multiple copies, as known for E. acidaminophilum [15] in contrast to C. sticklandii [12]. An open reading frame (ORF) known as grdX preceded the thioredoxin/glycine reductase gene cluster in C. sticklandii and is cotranscribed. The deduced 119 amino acid sequence shows low similarity values to an ORF also present in E. acidaminophilum and Thermoanaerobacter tengcogensis, but no known functional motif can be deduced. The genome of C. difficile contains both glycine reductase and sarcosine reductase, but no homolog of grdX. Therefore, grdX might be non-essential. www.sciencedirect.com Reactions involving the substrate-binding protein B Protein B represents the major problem for obtaining enzymatic active preparations of the reductases, probably due to the sensitivity of the carbonyl group of the GrdB protein that is responsible for substrate binding [9,15]. Previously, assays required large amounts of protein and took a long time [18,19]. The use of a partly heterologous complementation system, lacking only the required specific protein B components, is a major improvement [9] as the use of borohydride-inactivated extracts [15]. The insensitivity of BR against inactivation by hydroxylamine, borohydride and low concentrations of oxygen allowed the first isolation of homogenous proteins as well as raising antibodies against protein B [9]. Using these antibodies, the GrdE/GrdG-derived subunits of GR and SR were detected being post-translationally processed to 27/25 and 22 kDa subunits [15,20]. The 22 kDa protein derives by cysteinolysis from the C-terminal part of GrdE/GrdG [16]. In contrast to the D-proline reductase [11], the putatively formed pyruvoyl group does not bind the substrate. The carbonyl group might be involved in keeping the actual glycine or sarcosinebinding carbonyl group of the 47 kDa GrdB protein active (e.g. by transamination) if the imino group (Figure 2) is accidentally reduced. A terminal location is quite advantageous for such a function. The structure of the substrate-binding carbonyl group of GrdB/GrdF is still unknown, although the protein can be labeled with its respective radioactive substrate or 3H-borohydride. Even the binding area within defined cleaved peptides could not be resolved by mass Current Opinion in Chemical Biology 2004, 8:454–461 456 Mechanisms Figure 2 (a) (b) – Se OH Se O – O Gly Gly NH N H O NH O O N Thr H2O OH H2O O NH Thr NH NH4 + COO H2O GrdA-CH2-COO COO H H H NH2 H2O – – GrdA – O H Se – H Se H + NH O – NH Gly Gly N N H N O O Thr O O N NH NH (d) H (c) Thr Current Opinion in Chemical Biology Hypothetic, autocatalytic formation of a (a,b) labile imidazolone compound (in some analogy to that of [23]). (c,d) A close proximity to the Sec of GrdB would facilitate the formation of a Se-carboxymethyl selenoether from glycine after Schiff-base formation between this new carbonyl group and glycine. See text for further details. spectrometry due to an apparent lability of that area forming many unpredictable products or adducts [20]. The involvement of Sec in the carbon transfers should be unquestionable for it is present in all protein Bs. However, its direct involvement has not been proven for GrdB in contrast to GrdA [21]. Substrate binding does not involve pyridoxal phosphate. A post-translational modification seems to be possible. Positioning of the substratebinding carbonyl group quite close to the Sec moiety would facilitate a takeover of the C2-unit. Therefore, a possible carbonyl group within the protein might be one of the following: 1. A formylglycine group from a serine or cysteine as observed for sulfatases [22]. 2. A methylidene-imidazolone-like adduct [23] in which, however, the autocatalytically formed carbonyl group would take part in catalysis (see Figure 2 for a potential product from the conserved -Thr-Ser-Thr-Sec-Gly sequence). Current Opinion in Chemical Biology 2004, 8:454–461 3. A carbonyl group at the ureido moiety of citrulline formed by peptidyl-arginine deiminase [24]. 4. An aldehyde generated from the terminal amino group of a peptide-bound lysine. Most of these anticipated post-translational modifications involve a further enzyme [22]. The gene for this enzyme can only be outside of the analyzed operon structures, except for an autocatalytic processing mechanism depicted in Figure 2. The Schiff base formed with glycine could be located quite close to the nucleophilic selenolate, thus facilitating the formation of a Se-carboxymethyl selenoether bound to GrdB. Such a structure seems to be fragile in accordance with the experienced inability to analyze it. The UxxCxxC motif is characteristic for protein B. As revealed now, the CxxC motif is involved in protecting the essential Sec moiety against an accidental oxidation to a selenenic acid (R-Se-OH) [25]. The selenoperoxirewww.sciencedirect.com Glycine reductase mechanism Andreesen 457 doxin present in E. acidaminophilum [26] is not responsible for removal of hydrogen peroxide. Instead, protein B becomes purified by monitoring dithiothreitol (DTT)dependent peroxidase activity [25]. However, this purified protein B fraction is inactive in the GR assay. All three GrdB- and GrdE-derived proteins co-purify and show only some cleavage of GrdB to 36 and 30 kDa peptides as observed before using the GR assays [9,15,25]. If GrdB and its U/C or C/A mutants (in the above-shown motif) are formed in E. coli, all expressed GrdB variants are excessively cleaved to 23 kDa peptides, leaving only a small part of the 47 kDa protein intact and partly active. Sec is important for the low peroxidase reaction observed, compared with the Cys or Ala variants. However, the heterologous expression of genes for selenoproteins represents a general problem, especially from a Gram-positive bacterium in a Gram-negative species such as E. coli [27–29]. This can now be partly overcome by coexpression of the selB and selC genes from E. acidaminophilum [30]. So far, no effective genetic system has been established for an organism containing a GR. This limits further studies. The beneficial role of the two cysteines in the peroxidase reaction points to their protective function by first forming a selanylsulfide plus water in case the Sec is oxidized to a selenenic acid. This mixed Se–S bridge might be shifted to a disulfide between both Cys to reconstitute a functional selenol group [25]. Extracts catalyse an NADPH-dependent peroxidase reaction, indicating an involvement of the thioredoxin system. Further addition of GrdA or PrpU to a system of highly enriched, heterologously expressed proteins is still insufficient for an NADPH-dependent peroxidase activity [25]. However, GrdA should interact with the UxxCxxC motif of GrdB due to the anticipated transfer of the Se-carboxymethyl selenoether from GrdB to GrdA (Figure 1). The CxxU motif of GrdA is redox active [31]. Thus, GrdA might integrate both functions at this GrdB motif. Heterologous coexpression of grdB and grdE leads to the formation of two not-cleaved proteins that show a strong protein–protein interaction. However, the U/C mutant is inactive in the peroxidase reaction. Again, no proteolytic cleavage (nor activity) of this GrdB mutant is observed if one of the two artificially derived GrdE peptides is coexpressed [25]. Thus, in vivo processing of GrdE stabilizes GrdB. Therefore, protein B is generally isolated as a complex of these three proteins independent from the purification method [9,15,25]. GrdE is processed in vivo to a 25 and 22 kDa product [15]. The 22 kDa protein is N-terminally blocked, but VSACDK is the starting amino acid sequence after treatment with o-phenylenediamine, corresponding to the derived sequence from the conserved gene sequence for E. acidaminophilum and C. sticklandii [12,15,16]. This www.sciencedirect.com indicates that the cleavage should be between an Asn and a Cys moiety. The latter should be converted analogous to serine to give the newly formed pyruvoyl group, as in certain decarboxylases [32]. The heterologously formed GrdE protein requires harsh conditions for an in vitro cleavage [16]. By the reducing conditions used, a lactyl group would be expected as a product from pyruvate. However, the sequences obtained started with the Val or Asp located just behind the first or second Cys, indicating an unexplained removal of the Cys carbons. Both Cys moieties are essential for observing a cleavage of GrdE in contrast to other conserved moieties [33]. Artificial constructs of the cleavage area with malE and trxA can be cleaved to the corresponding products if an area of at least 30 amino acids from GrdE is in between. No in vitro cleavage is observed using an NADPH-generating system plus the thioredoxin system and GrdA. Reactions catalyzed by protein A (GrdA) Protein A integrates many essential functions although it is a small acidic 17 kDa protein that actually lacks a glycosylation [34]. As noted before, it should first accept the Se-carboxymethyl selenoether from GrdB (as likewise from GrdF and GrdH of SR and BR, respectively) (Figure 1), forming a common selenoether on this cytoplasmic, diffusible [35,36] and redox-active protein [31]. Its unique TECFVUTA/S motif is preceded at a distance of 16 amino acids by another conserved Cys of unknown function [14]. GrdA can be purified together with the thioredoxin reductase [37]. Its close interaction with these proteins is also evident by stimulating an electron transfer from DTT to NADPH [31]. In vivo, the reduction of oxidized GrdA will be catalyzed by the thioredoxin system. Only thioredoxins of bacteria with a GR contain a unique modified sequence at the otherwise strongly conserved WCGPCK motif, replacing the bulky Trp by a small amino acid such as Gly or Ser, whereas Val or Glu replaces Gly inside the redox-active motif. These modified thioredoxins do not interact with the thioredoxin reductase of E. coli, nor do they bind to weak anion exchange columns [37], indicating an important change in this otherwise promiscuous protein family that might be due to its interaction with the more voluminous selenium atom in GrdA compared with sulfur-containing proteins. The most unique reaction catalyzed by GR is the transfer of the GrdA-bound Se-carboxymethyl selenoether to an acetyl thioester at protein C [19,21,38,39]. The protein C preparations used were not homogeneous (as stated), and its subunit structure was revealed later [40]. The formation of an acetyl moiety from a Se-carboxymethyl selenoether implies a reduction reaction at the C2-unit coupled to an oxidation of the selenium in Sec to a mixed selanylsulfide at GrdA. Some possible transformation steps are given in Figure 3. At least, a protein C bound acetyl thioester is an established product of this Current Opinion in Chemical Biology 2004, 8:454–461 458 Mechanisms Figure 3 (a) (b) (c) S– S– O A Se-CH2-C Se- CH2-C OH -C SH O A –S O A Se- CH2-C OH OH H2O S S A + [CH2 =C=O] A OH Se- CH2 -C Se S-C SH OH A Se- CH2 -C OH H2O S C -SH A H2O SH –S -C Se- CH2 -C S-C A Se- CH2-C O S O C -S-C-CH3 O S O + A Se OH C -S-C-CH3 O + A C -S-C-CH3 Se Current Opinion in Chemical Biology Possible transformation steps involved in the formation of protein C-bound acetyl ester [38] from the GrdA-bound Se-carboxymethyl selenoether [21]. See text for further details. transformation [38,39]; thus, a conserved Cys residue at GrdC or GrdD should be the acetyl carrier. Because the smaller 40 kDa GrdD protein catalyses the final release of acetyl phosphate [17,40], the larger 54 kDa GrdC protein should be the partner for accepting the C2-unit from GrdA. One mechanism (Figure 3a) would involve a keten-like intermediate [41], which requires the total absence or immediate removal of water. In a second mechanism (Figure 3b), a nucleophilic attack by one of the two conserved Cys of GrdA might form an intramolecular adduct to the carbonyl group of the Se-carboxymethyl selenoether, which should split off water. This ‘caged’ C2-unit would consist of both a selenoether and a thioester. The latter would be displaced by a Cys of GrdC, whereas concomitantly the selenoether bond is reductively cleaved by forming a selanylsulfide with probably the Cys being adjacent to Sec. At least, this fact facilitates its later reduction by the thioredoxin system. A third possible mechanism (Figure 3c) has the same advantages as the second mechanism and similar reactions are proposed; however, the larger GrdC will initially catalyze the nucleophilic attack by a thiolate and the removal of water. The construction and heterologous expression of a GrdA triple mutant (the two Cys were replaced by Ala, Sec by Cys to improve the stability) did Current Opinion in Chemical Biology 2004, 8:454–461 not show a larger-sized stable adduct with GrdC (proposed according to Figure 3c) if this GrdA variant was carboxymethylated by 14C-iodoacetate. A corresponding GrdA/GrdC adduct could not be discriminated because the GrdA formed, in all cases, a stable and large complex with GrdC that might mask a covalently linked species (J Jäger and JR Andreesen, unpublished data). Reactions catalyzed by protein C Protein C is generally composed of the two subunits GrdC and GrdD, which form a very tight complex and show a strong association/dissociation behaviour in C. sticklandii, E. acidaminophilum and T. creatinophila, giving masses of 200–240 kDa and multimers [8,18,40]. By SDS gel electrophoresis, the determined molecular masses are much higher than deduced from the corresponding gene sequences, being 54 and 40 kDa [12,17]. Thus, an a2b2 structure is the minimal native form. GrdC has been sequenced from five organisms, three of them by genome analysis. The deduced similarities are for both subunits between 65% identity for C. difficile and about 55% identity for T. denticola. GrdC contains four conserved Cys at positions 172, 223, 228 and 261, all positioned in conserved areas. Cys223 www.sciencedirect.com Glycine reductase mechanism Andreesen 459 and 228 are surrounded by many acidic amino acids (ECSEEACGD), whereas RGFCAGP are conserved around Cys261. GrdC contains motifs that are also present in b-ketoacyl-carrier protein synthase III (FabH of E. coli) of different Gram-positive and Gram-negative bacteria [17], pointing to an involvement in transferring an acetyl group. GrdD generally forms a very tight complex with GrdC. Only GrdD could be isolated as individual protein from E. acidaminophilum, demonstrating its in vivo function in releasing acetyl phosphate from the acetyl thioester [40]. It contains two Cys of which Cys98 is inaccessible to chemical modifications. By contrast, Cys359 can be protected by adding acetyl phosphate. The corresponding C359A mutant is enzymatically inactive in the assay used [42], pointing to Cys359 as an essential thiol [17] from which the acetyl thioester is phosphorolytically liberated as acetyl phosphate. Conventional isolation of GrdC without GrdD proved to be impossible, and very substantial losses are generally obtained during final purification of protein C [8,40]. Atomic absorption analysis revealed a significant content of zinc in Protein C that is much lower (0 to 0.2 g atoms) if each subunit is individually expressed in E. coli [43]. Dialysis in the presence of zinc ions does not change these values, whereas an addition of zinc ions to the medium of E. coli increases these values to 1 and 0.4 g atoms per GrdC and GrdD, respectively. Coexpression of grdC and grdD results in a zinc content of 1 g atom per protomer. The purified GrdD contains a very low content (0.07 g atom), thus leaving the bulk of zinc ions (0.93 g atom) to GrdC [43]. Figure 4 depicts a hypothetical role of the mentioned Cys moieties and of a zinc ion in GrdC during the conversion of the GrdA-bound Se-carboxymethyl selenoether to a GrdC-bound acetyl ester. Cys223 and Cys228 seem to form a zinc-binding site, together with ligands such as His [44] and, thus, Zn2+ could also act as an electrophile for the pairs of free electrons at the selenium within the ether. An acidic and a basic amino acid might promote a nucleophilic attack of Cys261 by allowing intermediate formation of a diol at the carboxyl carbon. The conserved Arg258 might act as base for the nearby Cys261 or for the Cys at GrdA. The latter Cys thiolate should be involved in the oxidation of GrdA, perhaps facilitated by the coordinated zinc. A selenoether is more suited for a reductive cleavage than a sulfur ether and should donate its electrons to the C2-unit, forming a methyl group. The coordination of water by zinc is known from the carboanhydrase reaction [44]. Thus, its presence might help to remove water and, thus, to pull the reaction towards the acetyl thioester formation bound to Cys261 of GrdC. The similarity to FabH proteins points to a secondary transfer of the acetyl group from GrdC to Cys359 of GrdD by a transacetylase reaction. This proposed mechanism is in accord with the observed loss of one 18O using carboxyl labeled glycine [19] as well as with an exchange of tritium atoms from [3H] H2O into acetyl phosphate [38]. No reverse reaction such as a specific stimulation of GrdC in the transfer/arsenolysis Figure 4 GrdC GrdC His His Cys228-SH Cys228-SH Zn2+ Zn2+ Cys223-SH Cys223-SH O Se Se-CH2-C GrdA S – – O HA OH S B GrdA H H CH2-C H S OH –A OH B S H B Cys261 B Cys261 Current Opinion in Chemical Biology Proposed involvement of three conserved Cys moieties and of Zn2+ ions of GrdC in the unique transfer of the C2-unit from the GrdA-bound Se-carboxymethyl selenoether to a GrdC-bound acetyl thioester. Note that the water, drawn as enclosed by dashed lines, represents a consecutive step after its removal from the ‘dashed’ diol. The zinc site might be involved in this reaction. The arrangement of the individual components is only ‘dictated’ by the two-dimensional graphic to show expressed ideas. See text for further details. www.sciencedirect.com Current Opinion in Chemical Biology 2004, 8:454–461 460 Mechanisms reaction by GrdD could be observed using acetyl phosphate [43]. Proteins interacting with the GR complex Besides the thioredoxin system, formate is an excellent electron donor for glycine reduction. Thus, formate represses the oxidation of glycine (as also catalyzed by E. acidaminophilum) in favour of GR [45]. The formate dehydrogenase (FDH) is extremely active and contains, besides one Sec and a tungstopterin, five Fe/S clusters in the catalytic subunit FdhA [27]. The two extra Cys present in FdhA might interact directly with one of the three reductase systems as supposed by the co-purification of FDH and SR or BR [9]. HymB, a subunit of the Fe-only hydrogenase, is also co-purified [9,27]. The glycine decarboxylase complex of E. acidaminophilum is very unusual in lacking the dihydrolipoamide dehydrogenase component [46], as is now substantiated by specific gene probes and sequencing of the operon of glycine decarboxylase (gcv) [47]. The gene encoding a potential redox-active protein with a CxxU motif (prpU) is located close to the gcv operon. Similarities exist to a 12 kDa selenoprotein of T. denticola [5] that is annotated as a thioredoxin. It might be speculated that PrpU transfers electrons directly from the glycine decarboxylase to the GR (similar to or via GrdA [31]) or to the potential redox active Sec/Cys motif of GrdB [25]. Conclusions The development of a genetic system for one of these GRcontaining anaerobic bacteria is required, as well as the stabilization of labile components to obtain a deeper understanding of the detailed mechanism of the GR reaction. Acknowledgements The work reported from our laboratory was supported by grants of the Deutsche Forschungsgemeinschaft. I thank Tina Parther, Michael Reuter, Jana Jäger, Brit Eversmann and Anja Poehlein for citations of their unpublished results, David Rauh and Kathrin Makdessi for help with the generation of figures. 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