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Stabilization and conformational isomerization of the cofactor during the catalysis in hydrolytic ALDHs
Chemico-Biological Interactions 178 (2009) 79–83
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Stabilization and conformational isomerization of the cofactor during the catalysis in hydrolytic ALDHs Franc¸ois Talfournier ∗ , Arnaud Pailot, Claire Stinès-Chaumeil, Guy Branlant UMR 7567 CNRS - Nancy Université, Maturation des ARN et Enzymologie Moléculaire, Faculté des Sciences et Techniques, BP 239, 54506 Vandoeuvre-lès-Nancy Cedex, France
a r t i c l e
i n f o
Article history: Received 18 September 2008 Received in revised form 27 October 2008 Accepted 28 October 2008 Available online 5 November 2008 Keywords: Aldehyde dehydrogenase Cofactor conformations Hydride transfer Thioacylenzyme intermediate Deacylation
a b s t r a c t Over the past 15 years, mechanistic and structural aspects were studied extensively for hydrolytic ALDHs. One the most striking feature of nearly all X-ray structures of binary ALDH–NAD(P)+ complexes is the great conformational flexibility of the NMN moiety of the NAD(P)+ , in particular of the nicotinamide ring. However, the fact that the acylation step is efficient in GAPN (non-phosphorylating glyceraldehyde-3phosphate dehydrogenase) from Streptococcus mutans and in other hydrolytic ALDHs implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary complex to allow an efficient and stereospecific hydride transfer. Another key aspect of the chemical mechanism of this ALDH family is the requirement for the reduced NMN (NMNH) to move away from the initial position of the NMN for adequate positioning and activation of the deacylating water molecule by invariant E268 for completion of the reaction. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the stabilization of the NMN moiety of the cofactor during the acylation step and to provide structural evidence of conformational isomerization of the cofactor during the catalytic cycle of hydrolytic ALDHs. The results presented here will be discussed for their relevance to the two-step catalytic mechanism and from an evolutionary viewpoint. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Non-phosphorylating aldehyde dehydrogenases (ALDH) belong to a superfamily of phylogenetically and structurally related enzymes that catalyze the NAD(P)-dependent oxidation of a wide variety of aldehydes to their corresponding non-activated or CoAactivated acids. They share a similar two-step mechanism, first with an acylation step common to both families, and then a deacylation step that differs in the nature of the acyl acceptor. The acylation step involves the formation of a covalent hemithioacetal intermediate via the nucleophilic attack of the catalytic C302 on the aldehydic function followed by the hydride transfer that leads to formation of a thioacylenzyme intermediate and NAD(P)H (the amino acid numbering used for the biochemical and structural data is that defined by Wang and Weiner [1]). Then, the deacylation step includes the nucleophilic attack of a water or a CoA molecule on the thioacylenzyme intermediate, thus leading
Abbreviations: ALDH, aldehyde dehydrogenase; d-G3P, d-glyceraldehyde3-phosphate; CoA, coenzyme A; GAPN, non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase; MSDH, methylmalonate semialdehyde dehydrogenase; NMN(H), nicotinamide mononucleotide oxidized form (reduced form). ∗ Corresponding author. Tel.: +33 3 83 68 43 12; fax: +33 3 83 68 43 07. E-mail address: [email protected] (F. Talfournier). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.10.045
to non-activated or CoA-activated acids, respectively (Scheme 1). Mechanistic aspects were studied extensively for hydrolytic ALDHs, and several invariant residues were shown to be critical for the chemical mechanism [1–4]. In addition, enzymatic and structural studies on the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, which catalyzes the irreversible oxidation of glyceraldehyde-3-phosphate (G3P) into 3-phosphoglycerate, and other ALDHs have highlighted the essential roles of: (i) an oxyanion hole composed of the side-chain of invariant N169 and the main chain nitrogen of C302 that allows an efficient hydride transfer without base-catalyst assistance [5–8], and (ii) invariant E2681 in the rate-limiting hydrolysis step through activation and orientation of the attacking water molecule [9]. The fact that the acylation step in GAPN from S. mutans, and also many other hydrolytic ALDHs, is not rate-limiting and is efficient implies several prerequisites for this step [9]. One of those relates on the optimal positioning of the nicotinamide ring of the cofactor towards the hemithioacetal within the ternary complex. A review of the literature indicates that the majority of the Xray structures of binary ALDH–NAD(P)+ complexes reveal a great
1 E268 is invariant among the hydrolytic ALDH family, but absent in the CoAdependent one.
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Scheme 1. Schematic representation of the catalytic mechanism of ALDHs.
conformational flexibility of the NMN moiety of the NAD(P)+ , in particular of the nicotinamide ring, due to the peculiar binding mode of the cofactor to the Rossmann fold (see e.g. [6,10,11]. The occurrence of different conformations of the NMN moiety was also supported by NMR studies [12]. However, once the hemithioacetal intermediate is formed, the NMN moiety must be positioned such that an efficient and stereospecific hydride transfer occurs towards C-4 of the nicotinamide ring. In addition, the hydrolytic ALDHs exhibit an ordered sequential mechanism in which NAD(P)H dissociates last [9,13]. However, in the conformation described to be suitable for hydride transfer, the NMN moiety blocks the way and prevents the catalytic E268 from playing its role in the deacylation step. Therefore, another key aspect of the chemical mechanism of this ALDH family is the requirement for the reduced NMN (NMNH) to move away from the initial position of the NMN for adequate positioning and activation of the deacylating water molecule by invariant E268 for completion of the reaction. 2. Critical role of invariant T244 in the positioning of the nicotinamide ring within the covalent ternary complex of hydrolytic ALDHs Two discrete conformations, compatible with the two-step catalytic mechanism, were described for class 1 and class 2 ALDHs. In the conformation described to be suitable for an efficient hydride transfer, the nicotinamide ring is positioned between the catalytic C302 and the -methyl group of the invariant T244. This suggested a major role of the -methyl group of T244 in the positioning of the nicotinamide ring relative to the transient hemithioacetal intermediate [5,6,14]. This hypothesis was also supported by the structural evidence that the T244 side-chain could be held in position through hydrogen-bond interaction between its -hydroxyl group and the -amino group of the highly conserved K178 (Fig. 1). Recently, we investigated the role of invariant T244 in the hydride transfer efficiency by comparing the kinetic properties of the wild-type enzyme to those of the T244S, T244V and K178A variants of the GAPN from S. mutans [15]. In a previous study, it was shown that acylation and deacylation steps could be kinetically resolved for the GAPN-catalyzed reaction and that deacylation was rate-limiting under steady-state conditions. It was also demonstrated that the rate-determining step within the deacylation was associated with hydrolysis, whereas hydride transfer was rate-determining in acylation [9]. Substitution of serine for T244 suppressed the presteady-state burst of NADPH production observed for the wild-type enzyme, indicating that the rate-limiting step was shifted to the acylation in the T244S GAPN. The acylation rate was decreased by a factor 2900 compared to the wild type. The fact that an isotope effect was observed for the T244S GAPN when d-[1-2 H]G3P was used as the substrate proved that hydride transfer was rate-determining within acylation. The drastic decrease of the efficiency of hydride transfer for
the T244S GAPN could be the consequence of changes occurring in the location and orientation of the nicotinamide ring relative to the hemithioacetal intermediate, within the ternary complex, thereby preventing an efficient hydride transfer. The three-dimensional structure of the binary complex T244S GAPN/NADP+ obtained by X-ray crystallography further supported this hypothesis. Inspection of this structure showed that the absence of the -methyl group led to a unique stabilized position of the pyridinium ring clearly distinct from that hypothesized for the hydride transfer conformation in hydrolytic ALDHs including GAPN (Fig. 2). Determination of the structure of a hemithioacetal intermediate is not possible because of its transient nature. If it is assumed that the nicotinamide conformation in the T244S hemithioacetal intermediate–NADP+ complex is the one observed in the crystal structure of the binary complex T244S GAPN–NADP+ , the poor efficiency of the hydride transfer could be explained by non-optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate. The 0.6-unit increase in pKapp of C302 compared with the wild-type, which is similar to the contribution of the positively charged nicotinamide ring in lowering the pKapp of C302 in the wild-type, can be also interpreted as the consequence of positioning of the nicotinamide ring relative to C302 slightly different from that in the wild-type acylating ternary complex. Therefore, the role of the methyl group of T244 in the wild-type would be to mainly allow the
Fig. 1. Schematic view of part of the NADP+ -binding domain in GAPN from S. mutans showing the positioning of the nicotinamide ring (hydride transfer conformation) between C302 and the -methyl group of invariant T244, each at a distance <3.5 Å. The hydrogen-bond between the -hydroxyl group of T244 and the -amino group of the highly conserved K178 is also shown.
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Fig. 2. (A) Superimposition of the active site regions of the apo-like wild-type enzyme (in cyan, from [11]) and of the T244S GAPN (in red, from [15]) from S. mutans. The conformation of the NMN moiety in the crystalline binary complex wild-type GAPN/NADP+ is the one described to be suitable for efficient hydride transfer [11,14]. (B) Superimposition of the active site regions of the apo-like wild-type enzyme (in grey, from [11]) and of the T244S GAPN (in black, from [15]) from S. mutans. The conformation of the NMN moiety in the crystalline binary complex wild-type GAPN/NADP+ is the one described to be suitable for efficient hydride transfer [11,14]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer. Additionally, the role of the K178/T244 hydrogen-bond interaction in the correct positioning of the -methyl group of T244 relative to the nicotinamide ring of the cofactor was assessed by the substitutions of valine for T244 and alanine for K178. It was shown that removing the -hydroxyl group of T244 drastically alters the catalytic properties of the enzyme, with a rate constant associated with the rate-limiting acylation step decreased
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250,000-fold and 86-fold compared with the wild-type and T244S GAPNs, respectively. The substitution of alanine for the counterpart residue K178 also appears to be informative and complementary to the studies carried out on T244S GAPN. In fact, the K178A and T244S GAPNs exhibited very similar catalytic properties. First, the rate-limiting step was associated with hydride transfer and a 25,000-fold decrease of the kcat value was observed. Secondly, the pKapp values for Cys302 were equivalent in both ternary complexes, i.e., 6.7 compared with 6.8. Therefore it was tempting to interpret these results in a similar way to that developed for the T244S GAPN. So, the disruption of the T244–K178 hydrogen-bond interaction by substituting either valine for T244 or alanine for K178 would alter the positioning of T244, in particular of its -methyl group and thus of the nicotinamide ring that could explain the very poor efficiency of the hydride transfer. The major feature that has emerged from these kinetic and structural studies lies in the critical contribution of the -methyl group of invariant T244 in the efficiency of the GAPN acylation step. Such a role is expected to be general for all the members of the hydrolytic ALDH family. Interestingly, in a concomitant work, Weiner and collaborators reported the site-specific alteration of the T244 residue in the human ALDH1 and these authors also proposed that this residue likely has a direct role in binding the coenzyme in a productive conformation for hydride transfer [16]. 3. Structural evidence for a conformational isomerization of the cofactor during the catalytic cycle of hydrolytic ALDHs As mentioned Section 1, the movement of the NMNH is a prerequisite for the deacylation. This brings up the problem of the characterization of its conformation. Previous X-ray crystallographic studies of class 1 and class 2 ALDHs have identified a conformation of the NMN moiety hypothesized to be suitable for deacylation [5,17]. By comparison to the so-called “hydride trans-
Fig. 3. A: (a) Superimposition of the noncovalent ternary complex (hydride transfer conformation, from [7]) and the thioacylenzyme intermediate (from [18]) of GAPN from S. mutans showing the cofactor conformation during the acylation and deacylation steps. The cofactor conformations are shown in stick representation with cyan and grey carbons for NADP+ and NADPH, respectively. The two conformations found for the NADPH, ConfA (NMNH more solvent exposed), and ConfB (NMNH more bulky) are included. (b) Superimposition of the two NADPH conformations (ConfA and ConfB with carbons colored grey, from [18]) with NADH as observed in the human ALDH2/NADH binary complex (with carbons colored cyan, from [14]). B: (a) Superimposition of the noncovalent ternary complex (hydride transfer conformation, from [7]) and the thioacylenzyme intermediate (from [18]) of GAPN from S. mutans showing the cofactor conformation during the acylation and deacylation steps. The cofactor conformations are shown in stick representation with NADP+ colored black and NADPH colored grey, respectively. The two conformations found for the NADPH, ConfA (NMNH more solvent exposed), and ConfB (NMNH more bulky) are included. (b) Superimposition of the two NADPH conformations (ConfA and ConfB colored grey, from [18]) with NADH as observed in the human ALDH2/NADH binary complex (colored black, from [14]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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fer” conformation, the NMN part adopts a contracted conformation with the nicotinamide ring moving away from the active site by rotations about the pyrophosphate bonds. In a more recent study, the crystal structures of wild-type and C302S human mitochondrial ALDHs, in binary complexes with NAD+ and NADH, have led the authors to assign a preferential conformation of the cofactor related to its redox state; i.e., NAD+ would adopt a conformation suitable for hydride transfer, whereas NADH would prefer a contracted conformation suitable for the deacylation process [14]. However, structural data on GAPN from Thermoproteus tenax in complex with its cosubstrate NAD+ or its inhibitor NADPH showed that both cofactors adopt a hydride-transfer conformation [18]. This result suggested that the redox state of the cofactor is not necessarily the key factor triggering this isomerization step. In a recent paper, we provided for the first time structural evidence for a conformational isomerization of the cofactor during the catalytic cycle of a hydrolytic ALDH [19]. For this purpose, the structure of a reaction intermediate of the E268A-GAPN from S. mutans was obtained by soaking the crystals of the binary E268AGAPN/NADP+ complex with its natural substrate (G3P). This was the first thioacylenzyme intermediate characterized thus far in the ALDH superfamily, but it also revealed that reduction of cofactor upon acylation led to an extensive motion of the NMNH moiety with a flip of the reduced nicotinamide away from the active site (Fig. 3A). The two conformations found for the NMNH likely correlate with the double conformation observed for the catalytic C302 (one conformation assigned the C302 side-chain covalently bonded to the substrate, the second one to the free C302 side-chain). More importantly, they strongly differ from that observed for the NMN moiety of NADP+ in the Michaelis complex C302S-GAPN/NADP+ /G3P [7] and from the “hydrolysis” conformation described by Perez-Miller and Hurley for the binary complex human ALDH2/NADH [14] (Fig. 3B). Moreover, in the GAPN thioacylenzyme intermediate the oxygen atom at C1 of the substrate interacts with the N169-ND2 atom, which is known to constitute, together with the catalytic C302 amide nitrogen, the oxyanion hole required not only for the hydride transfer but also for the stabilization of the different intermediates during the catalytic cycle (through hydrogen-bonding with the C1 oxygen atom). In contrast, in the human ALDH2 binary complex, the oxygen atom of the amidic group from the reduced nicotinamide (NO7 atom) is postulated to be hydrogen-bonded to the N169 side-chain [14]. This would likely preclude N169 to play its role in the deacylation step. Moreover, in our case, reduction of the cofactor has occurred within the crystals in the presence of the natural substrate. Therefore, we proposed that this structure constitutes a reasonable picture of the thioacylenzyme intermediate for the physiological reaction. In that context, the conformation of the NADH described for the human ALDH2 could reflect an early stage of the conformational isomerization process that the cofactor undergoes after acylation. The fact that this conformational isomerization positions the NMNH moiety in a cavity which is present in other hydrolytic ALDH and involves conserved residues led us to propose that (i) it constitutes the pocket occupied by the NMNH moiety during the deacylation for all the members of the hydrolytic ALDH family and (ii) it might constitute the exit door for NAD(P)H as well.
4. Conclusions and open questions The studies on T244 and K178 mutated GAPNs support a critical role of the invariant T244 in the formation of a catalytically competent hemithioacetal/NAD(P)+ complex [15]. Such a role is probably general for the whole hydrolytic ALDH family. In addition to T244, the side-chain of invariant E399 was shown to play an essential role
by anchoring the NMN ribose through hydrogen-bonds with the hydroxyl groups of the ribose [20]. We also hypothesized that the negative charge of the tetrahedral oxyanion intermediate, which is never protonated within the active site, participates in the efficient positioning of the positively charged nicotinamide ring within the ternary complex hemithioacetal intermediate–NAD(P) [19]. Taken together, this probably delineates a pattern of interactions that stabilizes an efficient ‘hydride transfer conformation’ of the NMN moiety of the cofactor, once the transient hemithioacetal intermediate is formed. As already mentioned, the reduction of the cofactor upon acylation leads to an extensive motion of the NMNH in a pocket that likely constitutes the exit door for NADPH and arguments were provided that the NMNH conformation observed in GAPN is the one suitable for the deacylation step within all the hydrolytic ALDH family [19]. Nonetheless, the question regarding the molecular factors and the driving force, which promote isomerization of the cofactor, remains to be addressed. The loss of the positive charge of the nicotinamidium ring and the nonplanar conformation of its reduced form likely participate in triggering the NMNH conformational isomerization process. Additional structural data suggested that a low energetic barrier exists between the conformations adopted by the NMN(H) moiety during the two-step catalytic cycle of the hydrolytic ALDHs [19]. Based on the structure of the crystalline binary complex T244S GAPN/NADP, we proposed that the -methyl group of T244 might also play a role in the efficiency of the NMNH isomerization [15]. First, it might favor a metastable conformation of the oxidized form of the nicotinamide ring through van der Waals contact and thus decrease the energetic barrier between the “hydride transfer” and the flipped conformation. Second, its presence would sterically constrain the nonplanar conformation of the reduced nicotinamide thus favoring the flipping mechanism. This latter hypothesis is supported by a recent paper of Weiner and collaborators describing the catalytic properties of the T244S human ALDH1, in which they proposed that this substitution would slow the isomerization of the reduced cofactor out of the active site [20]. From an evolutionary viewpoint, the question arises of whether the pattern of interactions described to stabilize an efficient ‘hydride transfer conformation’ of the NMN moiety of the cofactor in the hydrolytic ALDHs is also operative in the members of the CoA-dependent family. In fact, the recent X-ray structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis, a CoA-dependent ALDH, in complex with NAD+ reveals a major difference in the stabilization mode of the NMN moiety, i.e., the nicotinamide ring is well defined in the electron density maps, in contrast to what is observed in nearly all X-ray structures of binary ALDH–NAD(P)+ complexes. This could originate mainly from a stabilization of the conformation of the nicotinamide ring through direct or indirect (via a water molecule) hydrogen-bonds between the carboxamide group and residues of the active site (unpublished data). Moreover, the T244 is replaced by an invariant valine residue in the MSDHs. However, one the two methyl groups of the -branched side-chain of V244 side-chain is positioned like that of -methyl of T244 and could fulfil a similar function thanks to the other -methyl group of V244 which could interact via hydrophobic interactions with the side-chain of the highly conserved M178. These hypotheses remain to be validated. Another question which has to be addressed relies on the occurrence of a cofactor isomerization after hydride transfer in the CoA-dependent ALDHs. Kinetic data recently published on the MSDH from B. subtilis are in favor of a ping-pong mechanism in which NADH release precedes the rate-limiting -decarboxylation process and the CoA-binding [21]. This is further supported by the X-ray structure of a thioacylenzyme intermediate recently obtained by soaking the crystals of the binary MSDH/NAD+ complex with the
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natural substrate methylmalonate semialdehyde which reveals the presence of a carboxylated covalent intermediate and the absence of cofactor in the electron density maps (unpublished data). Moreover, the pocket that is postulated to constitute the exit door of the reduced cofactor in the hydrolytic ALDHs is conserved and thus can play a similar role in the MSDH. From these results, it clearly appears that despite similar 3D structures and catalytic mechanisms, evolution has led to different strategies to achieve a similar two-step reaction. In that context, the molecular and structural factors responsible of the early release of the reduced cofactor in the CoA-dependent family remain to be identified. Acknowledgements We thank the members of the Biocrystallography team (UMR 7036 CNRS-Nancy Université) for fruitful collaboration. This research was supported by the CNRS, the French Ministère de la Recherche et de l’Enseignement Supérieur, the Nancy Université, the Institut Fédératif de Recherche 111 Bioingénierie and local funds from the Région Lorraine. Arnaud Pailot and Claire Stinès-Chaumeil gratefully thank the French Ministry of Research for financial support. References [1] X. Wang, H. Weiner, Involvement of glutamate 268 in the active site of human liver mitochondrial (class 2) aldehyde dehydrogenase as probed by site-directed mutagenesis, Biochemistry 34 (1995) 237–243. [2] J. Farres, T.T. Wang, S.J. Cunningham, H. Weiner, Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by sitedirected mutagenesis, Biochemistry 34 (1995) 2592–2598. [3] M. Vedadi, R. Szittner, L. Smillie, E. Meighen, Involvement of cysteine 289 in the catalytic activity of an NADP(+)-specific fatty aldehyde dehydrogenase from Vibrio harveyi, Biochemistry 34 (1995) 16725–16732. [4] M. Vedadi, E. Meighen, Critical glutamic acid residues affecting the mechanism and nucleotide specificity of Vibrio harveyi aldehyde dehydrogenase, Eur. J. Biochem. 246 (1997) 698–704. [5] C.G. Steinmetz, P. Xie, H. Weiner, D.T. Hurley, Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion, Structure (Lond.) 5 (1997) 701–711. [6] K. Johansson, M. El-Ahmad, S. Ramaswamy, L. Hjelmqvist, H. Jörnvall, H. Eklund, Structure of betaine aldehyde dehydrogenase at 2.1 A resolution, Protein Sci. 7 (1998) 2106–2117.
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[7] D. Cobessi, F. Tête Favier, S. Marchal, G. Branlant, A. Aubry, Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans, J. Mol. Biol. 300 (2000) 141–152. [8] S. Marchal, D. Cobessi, S. Rahuel-Clermont, F. Tete-Favier, A. Aubry, G. Branlant, Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans, Chem.-Biol. Interact. 130–132 (2001) 15–28. [9] S. Marchal, S. Rahuel-Clermont, G. Branlant, Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, Biochemistry 39 (2000) 3327–3335. [10] Z.J. Liu, Y.J. Sun, J. Rose, Y.J. Chung, C.D. Hsiao, W.R. Chang, I. Kuo, J. Perozich, R. Lindahl, J. Hempel, B.C. Wang, The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold, Nat. Struct. Biol. 4 (1997) 317–326. [11] D. Cobessi, F. Tête Favier, S. Marchal, S. Azza, G. Branlant, A. Aubry, Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans, J. Mol. Biol. 290 (1999) 161–173. [12] P.K. Hammen, A. Allali-Hassani, K. Hallenga, T.D. Hurley, H. Weiner, Multiple conformations of NAD and NADH when bound to human cytosolic and mitochondrial aldehyde dehydrogenase, Biochemistry 41 (2002) 7156–7168. [13] R.C. Vallari, R. Pietruszko, Kinetic mechanism of the human cytoplasmic aldehyde dehydrogenase E1, Arch. Biochem. Biophys. 212 (1981) 9–19. [14] S.J. Perez-Miller, T.D. Hurley, Coenzyme isomerization is integral to catalysis in aldehyde dehydrogenase, Biochemistry 42 (2003) 7100–7109. [15] A. Pailot, K. D’Ambrosio, C. Corbier, F. Talfournier, G. Branlant, Invariant Thr244 is essential for the efficient acylation step of the nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, Biochem. J. 400 (2006) 521–530. [16] K.K. Ho, T.D. Hurley, H. Weiner, Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis, Biochemistry 45 (2006) 9445–9453. [17] S.A. Moore, H.M. Baker, T.J. Blythe, K.E. Kitson, T.M. Kitson, E.N. Baker, Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases, Structure 6 (1998) 1541–1551. [18] E. Lorentzen, R. Hensel, T. Knura, H. Ahmed, E. Pohl, Structural basis of allosteric regulation and substrate specificity of the non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Thermoproteus tenax, J. Mol. Biol. 341 (2004) 815–828. [19] K. D’Ambrosio, A. Pailot, F. Talfournier, C. Didierjean, E. Benedetti, A. Aubry, G. Branlant, C. Corbier, The first crystal structure of a thioacylenzyme intermediate in the ALDH family: new coenzyme conformation and relevance to catalysis, Biochemistry 45 (2006) 2978–2986. [20] L. Ni, S. Sheikh, H. Weiner, Involvement of glutamate 399 and lysine 192 in the mechanism of human liver mitochondrial aldehyde dehydrogenase, J. Biol. Chem. 272 (1997) 18823–18826. [21] C. Stines-Chaumeil, F. Talfournier, G. Branlant, Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis, Biochem. J. 395 (2006) 107–115.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Stabilization and conformational isomerization of the cofactor during the catalysis in hydrolytic ALDHs Franc¸ois Talfournier ∗ , Arnaud Pailot, Claire Stinès-Chaumeil, Guy Branlant UMR 7567 CNRS - Nancy Université, Maturation des ARN et Enzymologie Moléculaire, Faculté des Sciences et Techniques, BP 239, 54506 Vandoeuvre-lès-Nancy Cedex, France
a r t i c l e
i n f o
Article history: Received 18 September 2008 Received in revised form 27 October 2008 Accepted 28 October 2008 Available online 5 November 2008 Keywords: Aldehyde dehydrogenase Cofactor conformations Hydride transfer Thioacylenzyme intermediate Deacylation
a b s t r a c t Over the past 15 years, mechanistic and structural aspects were studied extensively for hydrolytic ALDHs. One the most striking feature of nearly all X-ray structures of binary ALDH–NAD(P)+ complexes is the great conformational flexibility of the NMN moiety of the NAD(P)+ , in particular of the nicotinamide ring. However, the fact that the acylation step is efficient in GAPN (non-phosphorylating glyceraldehyde-3phosphate dehydrogenase) from Streptococcus mutans and in other hydrolytic ALDHs implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary complex to allow an efficient and stereospecific hydride transfer. Another key aspect of the chemical mechanism of this ALDH family is the requirement for the reduced NMN (NMNH) to move away from the initial position of the NMN for adequate positioning and activation of the deacylating water molecule by invariant E268 for completion of the reaction. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the stabilization of the NMN moiety of the cofactor during the acylation step and to provide structural evidence of conformational isomerization of the cofactor during the catalytic cycle of hydrolytic ALDHs. The results presented here will be discussed for their relevance to the two-step catalytic mechanism and from an evolutionary viewpoint. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Non-phosphorylating aldehyde dehydrogenases (ALDH) belong to a superfamily of phylogenetically and structurally related enzymes that catalyze the NAD(P)-dependent oxidation of a wide variety of aldehydes to their corresponding non-activated or CoAactivated acids. They share a similar two-step mechanism, first with an acylation step common to both families, and then a deacylation step that differs in the nature of the acyl acceptor. The acylation step involves the formation of a covalent hemithioacetal intermediate via the nucleophilic attack of the catalytic C302 on the aldehydic function followed by the hydride transfer that leads to formation of a thioacylenzyme intermediate and NAD(P)H (the amino acid numbering used for the biochemical and structural data is that defined by Wang and Weiner [1]). Then, the deacylation step includes the nucleophilic attack of a water or a CoA molecule on the thioacylenzyme intermediate, thus leading
Abbreviations: ALDH, aldehyde dehydrogenase; d-G3P, d-glyceraldehyde3-phosphate; CoA, coenzyme A; GAPN, non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase; MSDH, methylmalonate semialdehyde dehydrogenase; NMN(H), nicotinamide mononucleotide oxidized form (reduced form). ∗ Corresponding author. Tel.: +33 3 83 68 43 12; fax: +33 3 83 68 43 07. E-mail address: [email protected] (F. Talfournier). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.10.045
to non-activated or CoA-activated acids, respectively (Scheme 1). Mechanistic aspects were studied extensively for hydrolytic ALDHs, and several invariant residues were shown to be critical for the chemical mechanism [1–4]. In addition, enzymatic and structural studies on the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, which catalyzes the irreversible oxidation of glyceraldehyde-3-phosphate (G3P) into 3-phosphoglycerate, and other ALDHs have highlighted the essential roles of: (i) an oxyanion hole composed of the side-chain of invariant N169 and the main chain nitrogen of C302 that allows an efficient hydride transfer without base-catalyst assistance [5–8], and (ii) invariant E2681 in the rate-limiting hydrolysis step through activation and orientation of the attacking water molecule [9]. The fact that the acylation step in GAPN from S. mutans, and also many other hydrolytic ALDHs, is not rate-limiting and is efficient implies several prerequisites for this step [9]. One of those relates on the optimal positioning of the nicotinamide ring of the cofactor towards the hemithioacetal within the ternary complex. A review of the literature indicates that the majority of the Xray structures of binary ALDH–NAD(P)+ complexes reveal a great
1 E268 is invariant among the hydrolytic ALDH family, but absent in the CoAdependent one.
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Scheme 1. Schematic representation of the catalytic mechanism of ALDHs.
conformational flexibility of the NMN moiety of the NAD(P)+ , in particular of the nicotinamide ring, due to the peculiar binding mode of the cofactor to the Rossmann fold (see e.g. [6,10,11]. The occurrence of different conformations of the NMN moiety was also supported by NMR studies [12]. However, once the hemithioacetal intermediate is formed, the NMN moiety must be positioned such that an efficient and stereospecific hydride transfer occurs towards C-4 of the nicotinamide ring. In addition, the hydrolytic ALDHs exhibit an ordered sequential mechanism in which NAD(P)H dissociates last [9,13]. However, in the conformation described to be suitable for hydride transfer, the NMN moiety blocks the way and prevents the catalytic E268 from playing its role in the deacylation step. Therefore, another key aspect of the chemical mechanism of this ALDH family is the requirement for the reduced NMN (NMNH) to move away from the initial position of the NMN for adequate positioning and activation of the deacylating water molecule by invariant E268 for completion of the reaction. 2. Critical role of invariant T244 in the positioning of the nicotinamide ring within the covalent ternary complex of hydrolytic ALDHs Two discrete conformations, compatible with the two-step catalytic mechanism, were described for class 1 and class 2 ALDHs. In the conformation described to be suitable for an efficient hydride transfer, the nicotinamide ring is positioned between the catalytic C302 and the -methyl group of the invariant T244. This suggested a major role of the -methyl group of T244 in the positioning of the nicotinamide ring relative to the transient hemithioacetal intermediate [5,6,14]. This hypothesis was also supported by the structural evidence that the T244 side-chain could be held in position through hydrogen-bond interaction between its -hydroxyl group and the -amino group of the highly conserved K178 (Fig. 1). Recently, we investigated the role of invariant T244 in the hydride transfer efficiency by comparing the kinetic properties of the wild-type enzyme to those of the T244S, T244V and K178A variants of the GAPN from S. mutans [15]. In a previous study, it was shown that acylation and deacylation steps could be kinetically resolved for the GAPN-catalyzed reaction and that deacylation was rate-limiting under steady-state conditions. It was also demonstrated that the rate-determining step within the deacylation was associated with hydrolysis, whereas hydride transfer was rate-determining in acylation [9]. Substitution of serine for T244 suppressed the presteady-state burst of NADPH production observed for the wild-type enzyme, indicating that the rate-limiting step was shifted to the acylation in the T244S GAPN. The acylation rate was decreased by a factor 2900 compared to the wild type. The fact that an isotope effect was observed for the T244S GAPN when d-[1-2 H]G3P was used as the substrate proved that hydride transfer was rate-determining within acylation. The drastic decrease of the efficiency of hydride transfer for
the T244S GAPN could be the consequence of changes occurring in the location and orientation of the nicotinamide ring relative to the hemithioacetal intermediate, within the ternary complex, thereby preventing an efficient hydride transfer. The three-dimensional structure of the binary complex T244S GAPN/NADP+ obtained by X-ray crystallography further supported this hypothesis. Inspection of this structure showed that the absence of the -methyl group led to a unique stabilized position of the pyridinium ring clearly distinct from that hypothesized for the hydride transfer conformation in hydrolytic ALDHs including GAPN (Fig. 2). Determination of the structure of a hemithioacetal intermediate is not possible because of its transient nature. If it is assumed that the nicotinamide conformation in the T244S hemithioacetal intermediate–NADP+ complex is the one observed in the crystal structure of the binary complex T244S GAPN–NADP+ , the poor efficiency of the hydride transfer could be explained by non-optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate. The 0.6-unit increase in pKapp of C302 compared with the wild-type, which is similar to the contribution of the positively charged nicotinamide ring in lowering the pKapp of C302 in the wild-type, can be also interpreted as the consequence of positioning of the nicotinamide ring relative to C302 slightly different from that in the wild-type acylating ternary complex. Therefore, the role of the methyl group of T244 in the wild-type would be to mainly allow the
Fig. 1. Schematic view of part of the NADP+ -binding domain in GAPN from S. mutans showing the positioning of the nicotinamide ring (hydride transfer conformation) between C302 and the -methyl group of invariant T244, each at a distance <3.5 Å. The hydrogen-bond between the -hydroxyl group of T244 and the -amino group of the highly conserved K178 is also shown.
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Fig. 2. (A) Superimposition of the active site regions of the apo-like wild-type enzyme (in cyan, from [11]) and of the T244S GAPN (in red, from [15]) from S. mutans. The conformation of the NMN moiety in the crystalline binary complex wild-type GAPN/NADP+ is the one described to be suitable for efficient hydride transfer [11,14]. (B) Superimposition of the active site regions of the apo-like wild-type enzyme (in grey, from [11]) and of the T244S GAPN (in black, from [15]) from S. mutans. The conformation of the NMN moiety in the crystalline binary complex wild-type GAPN/NADP+ is the one described to be suitable for efficient hydride transfer [11,14]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer. Additionally, the role of the K178/T244 hydrogen-bond interaction in the correct positioning of the -methyl group of T244 relative to the nicotinamide ring of the cofactor was assessed by the substitutions of valine for T244 and alanine for K178. It was shown that removing the -hydroxyl group of T244 drastically alters the catalytic properties of the enzyme, with a rate constant associated with the rate-limiting acylation step decreased
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250,000-fold and 86-fold compared with the wild-type and T244S GAPNs, respectively. The substitution of alanine for the counterpart residue K178 also appears to be informative and complementary to the studies carried out on T244S GAPN. In fact, the K178A and T244S GAPNs exhibited very similar catalytic properties. First, the rate-limiting step was associated with hydride transfer and a 25,000-fold decrease of the kcat value was observed. Secondly, the pKapp values for Cys302 were equivalent in both ternary complexes, i.e., 6.7 compared with 6.8. Therefore it was tempting to interpret these results in a similar way to that developed for the T244S GAPN. So, the disruption of the T244–K178 hydrogen-bond interaction by substituting either valine for T244 or alanine for K178 would alter the positioning of T244, in particular of its -methyl group and thus of the nicotinamide ring that could explain the very poor efficiency of the hydride transfer. The major feature that has emerged from these kinetic and structural studies lies in the critical contribution of the -methyl group of invariant T244 in the efficiency of the GAPN acylation step. Such a role is expected to be general for all the members of the hydrolytic ALDH family. Interestingly, in a concomitant work, Weiner and collaborators reported the site-specific alteration of the T244 residue in the human ALDH1 and these authors also proposed that this residue likely has a direct role in binding the coenzyme in a productive conformation for hydride transfer [16]. 3. Structural evidence for a conformational isomerization of the cofactor during the catalytic cycle of hydrolytic ALDHs As mentioned Section 1, the movement of the NMNH is a prerequisite for the deacylation. This brings up the problem of the characterization of its conformation. Previous X-ray crystallographic studies of class 1 and class 2 ALDHs have identified a conformation of the NMN moiety hypothesized to be suitable for deacylation [5,17]. By comparison to the so-called “hydride trans-
Fig. 3. A: (a) Superimposition of the noncovalent ternary complex (hydride transfer conformation, from [7]) and the thioacylenzyme intermediate (from [18]) of GAPN from S. mutans showing the cofactor conformation during the acylation and deacylation steps. The cofactor conformations are shown in stick representation with cyan and grey carbons for NADP+ and NADPH, respectively. The two conformations found for the NADPH, ConfA (NMNH more solvent exposed), and ConfB (NMNH more bulky) are included. (b) Superimposition of the two NADPH conformations (ConfA and ConfB with carbons colored grey, from [18]) with NADH as observed in the human ALDH2/NADH binary complex (with carbons colored cyan, from [14]). B: (a) Superimposition of the noncovalent ternary complex (hydride transfer conformation, from [7]) and the thioacylenzyme intermediate (from [18]) of GAPN from S. mutans showing the cofactor conformation during the acylation and deacylation steps. The cofactor conformations are shown in stick representation with NADP+ colored black and NADPH colored grey, respectively. The two conformations found for the NADPH, ConfA (NMNH more solvent exposed), and ConfB (NMNH more bulky) are included. (b) Superimposition of the two NADPH conformations (ConfA and ConfB colored grey, from [18]) with NADH as observed in the human ALDH2/NADH binary complex (colored black, from [14]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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fer” conformation, the NMN part adopts a contracted conformation with the nicotinamide ring moving away from the active site by rotations about the pyrophosphate bonds. In a more recent study, the crystal structures of wild-type and C302S human mitochondrial ALDHs, in binary complexes with NAD+ and NADH, have led the authors to assign a preferential conformation of the cofactor related to its redox state; i.e., NAD+ would adopt a conformation suitable for hydride transfer, whereas NADH would prefer a contracted conformation suitable for the deacylation process [14]. However, structural data on GAPN from Thermoproteus tenax in complex with its cosubstrate NAD+ or its inhibitor NADPH showed that both cofactors adopt a hydride-transfer conformation [18]. This result suggested that the redox state of the cofactor is not necessarily the key factor triggering this isomerization step. In a recent paper, we provided for the first time structural evidence for a conformational isomerization of the cofactor during the catalytic cycle of a hydrolytic ALDH [19]. For this purpose, the structure of a reaction intermediate of the E268A-GAPN from S. mutans was obtained by soaking the crystals of the binary E268AGAPN/NADP+ complex with its natural substrate (G3P). This was the first thioacylenzyme intermediate characterized thus far in the ALDH superfamily, but it also revealed that reduction of cofactor upon acylation led to an extensive motion of the NMNH moiety with a flip of the reduced nicotinamide away from the active site (Fig. 3A). The two conformations found for the NMNH likely correlate with the double conformation observed for the catalytic C302 (one conformation assigned the C302 side-chain covalently bonded to the substrate, the second one to the free C302 side-chain). More importantly, they strongly differ from that observed for the NMN moiety of NADP+ in the Michaelis complex C302S-GAPN/NADP+ /G3P [7] and from the “hydrolysis” conformation described by Perez-Miller and Hurley for the binary complex human ALDH2/NADH [14] (Fig. 3B). Moreover, in the GAPN thioacylenzyme intermediate the oxygen atom at C1 of the substrate interacts with the N169-ND2 atom, which is known to constitute, together with the catalytic C302 amide nitrogen, the oxyanion hole required not only for the hydride transfer but also for the stabilization of the different intermediates during the catalytic cycle (through hydrogen-bonding with the C1 oxygen atom). In contrast, in the human ALDH2 binary complex, the oxygen atom of the amidic group from the reduced nicotinamide (NO7 atom) is postulated to be hydrogen-bonded to the N169 side-chain [14]. This would likely preclude N169 to play its role in the deacylation step. Moreover, in our case, reduction of the cofactor has occurred within the crystals in the presence of the natural substrate. Therefore, we proposed that this structure constitutes a reasonable picture of the thioacylenzyme intermediate for the physiological reaction. In that context, the conformation of the NADH described for the human ALDH2 could reflect an early stage of the conformational isomerization process that the cofactor undergoes after acylation. The fact that this conformational isomerization positions the NMNH moiety in a cavity which is present in other hydrolytic ALDH and involves conserved residues led us to propose that (i) it constitutes the pocket occupied by the NMNH moiety during the deacylation for all the members of the hydrolytic ALDH family and (ii) it might constitute the exit door for NAD(P)H as well.
4. Conclusions and open questions The studies on T244 and K178 mutated GAPNs support a critical role of the invariant T244 in the formation of a catalytically competent hemithioacetal/NAD(P)+ complex [15]. Such a role is probably general for the whole hydrolytic ALDH family. In addition to T244, the side-chain of invariant E399 was shown to play an essential role
by anchoring the NMN ribose through hydrogen-bonds with the hydroxyl groups of the ribose [20]. We also hypothesized that the negative charge of the tetrahedral oxyanion intermediate, which is never protonated within the active site, participates in the efficient positioning of the positively charged nicotinamide ring within the ternary complex hemithioacetal intermediate–NAD(P) [19]. Taken together, this probably delineates a pattern of interactions that stabilizes an efficient ‘hydride transfer conformation’ of the NMN moiety of the cofactor, once the transient hemithioacetal intermediate is formed. As already mentioned, the reduction of the cofactor upon acylation leads to an extensive motion of the NMNH in a pocket that likely constitutes the exit door for NADPH and arguments were provided that the NMNH conformation observed in GAPN is the one suitable for the deacylation step within all the hydrolytic ALDH family [19]. Nonetheless, the question regarding the molecular factors and the driving force, which promote isomerization of the cofactor, remains to be addressed. The loss of the positive charge of the nicotinamidium ring and the nonplanar conformation of its reduced form likely participate in triggering the NMNH conformational isomerization process. Additional structural data suggested that a low energetic barrier exists between the conformations adopted by the NMN(H) moiety during the two-step catalytic cycle of the hydrolytic ALDHs [19]. Based on the structure of the crystalline binary complex T244S GAPN/NADP, we proposed that the -methyl group of T244 might also play a role in the efficiency of the NMNH isomerization [15]. First, it might favor a metastable conformation of the oxidized form of the nicotinamide ring through van der Waals contact and thus decrease the energetic barrier between the “hydride transfer” and the flipped conformation. Second, its presence would sterically constrain the nonplanar conformation of the reduced nicotinamide thus favoring the flipping mechanism. This latter hypothesis is supported by a recent paper of Weiner and collaborators describing the catalytic properties of the T244S human ALDH1, in which they proposed that this substitution would slow the isomerization of the reduced cofactor out of the active site [20]. From an evolutionary viewpoint, the question arises of whether the pattern of interactions described to stabilize an efficient ‘hydride transfer conformation’ of the NMN moiety of the cofactor in the hydrolytic ALDHs is also operative in the members of the CoA-dependent family. In fact, the recent X-ray structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis, a CoA-dependent ALDH, in complex with NAD+ reveals a major difference in the stabilization mode of the NMN moiety, i.e., the nicotinamide ring is well defined in the electron density maps, in contrast to what is observed in nearly all X-ray structures of binary ALDH–NAD(P)+ complexes. This could originate mainly from a stabilization of the conformation of the nicotinamide ring through direct or indirect (via a water molecule) hydrogen-bonds between the carboxamide group and residues of the active site (unpublished data). Moreover, the T244 is replaced by an invariant valine residue in the MSDHs. However, one the two methyl groups of the -branched side-chain of V244 side-chain is positioned like that of -methyl of T244 and could fulfil a similar function thanks to the other -methyl group of V244 which could interact via hydrophobic interactions with the side-chain of the highly conserved M178. These hypotheses remain to be validated. Another question which has to be addressed relies on the occurrence of a cofactor isomerization after hydride transfer in the CoA-dependent ALDHs. Kinetic data recently published on the MSDH from B. subtilis are in favor of a ping-pong mechanism in which NADH release precedes the rate-limiting -decarboxylation process and the CoA-binding [21]. This is further supported by the X-ray structure of a thioacylenzyme intermediate recently obtained by soaking the crystals of the binary MSDH/NAD+ complex with the
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natural substrate methylmalonate semialdehyde which reveals the presence of a carboxylated covalent intermediate and the absence of cofactor in the electron density maps (unpublished data). Moreover, the pocket that is postulated to constitute the exit door of the reduced cofactor in the hydrolytic ALDHs is conserved and thus can play a similar role in the MSDH. From these results, it clearly appears that despite similar 3D structures and catalytic mechanisms, evolution has led to different strategies to achieve a similar two-step reaction. In that context, the molecular and structural factors responsible of the early release of the reduced cofactor in the CoA-dependent family remain to be identified. Acknowledgements We thank the members of the Biocrystallography team (UMR 7036 CNRS-Nancy Université) for fruitful collaboration. This research was supported by the CNRS, the French Ministère de la Recherche et de l’Enseignement Supérieur, the Nancy Université, the Institut Fédératif de Recherche 111 Bioingénierie and local funds from the Région Lorraine. Arnaud Pailot and Claire Stinès-Chaumeil gratefully thank the French Ministry of Research for financial support. References [1] X. Wang, H. Weiner, Involvement of glutamate 268 in the active site of human liver mitochondrial (class 2) aldehyde dehydrogenase as probed by site-directed mutagenesis, Biochemistry 34 (1995) 237–243. [2] J. Farres, T.T. Wang, S.J. Cunningham, H. Weiner, Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by sitedirected mutagenesis, Biochemistry 34 (1995) 2592–2598. [3] M. Vedadi, R. Szittner, L. Smillie, E. Meighen, Involvement of cysteine 289 in the catalytic activity of an NADP(+)-specific fatty aldehyde dehydrogenase from Vibrio harveyi, Biochemistry 34 (1995) 16725–16732. [4] M. Vedadi, E. Meighen, Critical glutamic acid residues affecting the mechanism and nucleotide specificity of Vibrio harveyi aldehyde dehydrogenase, Eur. J. Biochem. 246 (1997) 698–704. [5] C.G. Steinmetz, P. Xie, H. Weiner, D.T. Hurley, Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion, Structure (Lond.) 5 (1997) 701–711. [6] K. Johansson, M. El-Ahmad, S. Ramaswamy, L. Hjelmqvist, H. Jörnvall, H. Eklund, Structure of betaine aldehyde dehydrogenase at 2.1 A resolution, Protein Sci. 7 (1998) 2106–2117.
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