FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1969727059> ?p ?o ?g. }

• W1969727059 endingPage "30556" @default.
• W1969727059 startingPage "30550" @default.
• W1969727059 abstract "Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 Å crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax. Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 Å crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax. Mitochondrial aldehyde dehydrogenase (ALDH2) 1The abbreviations used are: ALDH2, mitochondrial aldehyde dehydrogenase; ALDH2*2, semidominant polymorphism of ALDH2; MCAD, medium chain acyl-CoA dehydrogenase; ACES, N-(2-Acetamido)-2-aminoethanesulfonic acid. is best known for its role in ethanol metabolism, oxidizing acetaldehyde to acetate (1Hurley T.D. Edenberg H.J. Li T.K. Licinio J. Wong M. Pharmacogenomics: The Search for Individualized Therapies. Wiley-VCH, Weinheim, Germany2002: 417-441Crossref Google Scholar). More recently, an additional role for ALDH2 has been described as the initiator of nitroglycerin bioactivation (2Chen Z. Zhang J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8306-8311Crossref PubMed Scopus (499) Google Scholar). About 40% of the East Asian population carries a semidominant polymorphism of the ALDH2 gene, ALDH2*2 (3Seitz H.K. Matsuzaki S. Yokoyama A. Homann N. Vakevainen S. Wang X.D. Alcohol. Clin. Exp. Res. 2001; 25: 137S-143SCrossref PubMed Scopus (130) Google Scholar). The associated glutamate to lysine substitution at position 487 causes the enzyme to be nearly inactive in vivo. The resulting phenotype is characterized by an aversive response to ethanol consumption, which may include facial flushing, nausea, and tachycardia, with more severe reactions observed in those individuals who are homozygous for the polymorphism (4Peng G.S. Wang M.F. Chen C.Y. Luu S.U. Chou H.C. Li T.K. Yin S.J. Pharmacogenetics. 1999; 9: 463-476PubMed Google Scholar). This adverse effect has been linked to a lower frequency of alcoholism among those both hetero- and homozygous for the variant enzyme. Even so, ALDH2*2 has been linked to alcoholic liver disease as well as orapharyngo-laryngeal and esophageal cancers in alcoholic patients (5Enomoto N. Takase S. Takada N. Takada A. Hepatology. 1991; 13: 1071-1075Crossref PubMed Scopus (132) Google Scholar, 6Yokoyama A. Muramatsu T. Omori T. Matsushita S. Yoshimizu H. Higuchi S. Yokoyama T. Maruyama K. Ishii H. Alcohol. Clin. Exp. Res. 1999; 23: 1705-1710Crossref PubMed Scopus (89) Google Scholar). The kinetic properties of human ALDH2*2 have been determined using protein expressed and purified from Escherichia coli (7Farres J. Wang X. Takahashi K. Cunningham S.J. Wang T.T. Weiner H. J. Biol. Chem. 1994; 269: 13854-13860Abstract Full Text PDF PubMed Google Scholar). The variant enzyme was found to be active, but it exhibits a 200-fold increased Km for NAD+ and a diminished kcat. The enzyme Km for NAD+ exceeds the available concentration in the cell by 15-fold, which, combined with the 10-fold lower kcat value, would lead to an approximately 100-fold lower activity in vivo (7Farres J. Wang X. Takahashi K. Cunningham S.J. Wang T.T. Weiner H. J. Biol. Chem. 1994; 269: 13854-13860Abstract Full Text PDF PubMed Google Scholar). The three-dimensional structure of ALDH2 has been solved and described previously (8Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure. 1997; 5: 701-711Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 9Ni L. Zhou J. Hurley T.D. Weiner H. Protein Sci. 1999; 8: 2784-2790Crossref PubMed Scopus (73) Google Scholar). The enzyme is a tetramer of four identical subunits, each consisting of 500 amino acid residues (Fig. 1). This tetramer can be regarded as a dimer of dimers in that the interface between monomers that form a dimer is different and more extensive than the interface between the two dimers that form the tetramer. Each subunit is composed of three main domains: the catalytic domain, the coenzyme or NAD+-binding domain, and the oligomerization domain (8Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure. 1997; 5: 701-711Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar) (Fig. 2). Glu487 is located within the oligomerization domain, which is important for both dimer and tetramer formation. In wild-type ALDH2, Glu487 forms hydrogen bonds with Arg264 of the same subunit and with Arg475 of the adjacent dimer partner. Consequently, the disruption of these interactions by the presence of Lys487 was projected to perturb the structure of both its own subunit as well as its dimer partner (8Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure. 1997; 5: 701-711Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The interaction across the dimer interface has been thought to be responsible for the dominant effects of this mutation (8Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure. 1997; 5: 701-711Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar).Fig. 2Subunits of ALDH2. a, wild-type subunit with its three major domains labeled. The secondary structure features are color-coded: navy blue, αF; gold, β10; red, αG; green, β11; orange, loop at residues 269-273; violet, loop at residues 463-478. Residues 264, 487, and 475, shown in a space-filled representation, are labeled and colored green, pink, and violet, respectively. The catalytic nucleophile, Cys302, is labeled and denoted by a black ribbon. b, an alignment of ALDH2*2 subunits A and B to wild type. In this dimer representation, the ALDH2*2 αG helix is shown in red, and wild type is shown in blue. The helix of ALDH2*2 shifts 3 Å into the NAD+-binding cleft. Residues 264, 487, and 475 from the wild-type structure are colored as in a.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to the dominance of ALDH2*2, other examples of intersubunit communication have been observed in ALDH2. Half-site reactivity for ALDH2 was first demonstrated in wild-type horse liver ALDH2 where there was a pre-steady-state burst of NADH formation equivalent to 2 mol of NADH per ALDH2 tetramer (10Weiner H. Hu J.H. Sanny C.G. J. Biol. Chem. 1976; 251: 3853-3855Abstract Full Text PDF PubMed Google Scholar). Wild-type rat ALDH2 demonstrated the same pre-steady-state burst, and the rate-limiting step was determined to be the deacylation step (7Farres J. Wang X. Takahashi K. Cunningham S.J. Wang T.T. Weiner H. J. Biol. Chem. 1994; 269: 13854-13860Abstract Full Text PDF PubMed Google Scholar). However, ALDH2*2 does not demonstrate the pre-steady-state burst of NADH formation, suggesting that for the variant enzyme, the rate-limiting step occurs before NADH formation, possibly involving thiohemiacetal formation (7Farres J. Wang X. Takahashi K. Cunningham S.J. Wang T.T. Weiner H. J. Biol. Chem. 1994; 269: 13854-13860Abstract Full Text PDF PubMed Google Scholar). Based on studies conducted with wild type-ALDH2*2 heterotetramers, the current model of half-site reactivity is one where only one subunit per dimer pair is active in wild-type ALDH2 (11Zhou J. Weiner H. Biochemistry. 2000; 39: 12019-12024Crossref PubMed Scopus (29) Google Scholar). Whether activity alternates between active sites or remains fixed in a particular active site is not yet understood. These kinetics studies support a model of dominance where a variant subunit would inactivate its wild-type dimer partner but would have no effect on the subunits in the dimer pair across the tetramer interface. Numerous aldehyde dehydrogenase crystal structures have shown that the nicotinamide portion of NAD(P) is flexible and occupies two main conformations (9Ni L. Zhou J. Hurley T.D. Weiner H. Protein Sci. 1999; 8: 2784-2790Crossref PubMed Scopus (73) Google Scholar, 12Perez-Miller S. Hurley T.D. Biochemistry. 2003; 42: 7100-7109Crossref PubMed Scopus (142) Google Scholar, 13Hammen P.K. Allali-Hassani A. Hallenga K. Hurley T.D. Weiner H. Biochemistry. 2002; 41: 7156-7168Crossref PubMed Scopus (72) Google Scholar, 14Moore S.A. Baker H.M. Blythe T.J. Kitson K.E. Kitson T.M. Baker E.N. Structure. 1998; 6: 1541-1551Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 15Hurley T. Perez-Miller S. Breen H. Chem. Biol. Interact. 2001; 130-132: 3-14Crossref PubMed Scopus (27) Google Scholar). These conformations are likely to play a functional role in catalysis where the extended conformation is primarily occupied by NAD+ and is conducive to hydride transfer, and the other, more contracted conformation is primarily occupied by NADH and is conducive to acyl-enzyme hydrolysis (12Perez-Miller S. Hurley T.D. Biochemistry. 2003; 42: 7100-7109Crossref PubMed Scopus (142) Google Scholar). Magnesium is important for stabilizing these two conformations, thereby elucidating its documented role in elevating Vmax. To further study the variant ALDH2 enzyme, we have solved the apoenzyme crystal structure of ALDH2*2 to 2.1 Å resolution. This structure reveals that mutation of Glu487 to lysine leads to extreme disorder of the dimer interface, including the helix αG that comprises part of the NAD+-binding site. The β-strand containing the general base Glu268 has shifted away from the active site, and other main-chain and side-chain shifts lead to an altered coenzyme-binding site. Lysine 487 itself, however, is well ordered, although the residue no longer contributes to favorable intersubunit interactions. Materials—Dithiothreitol, DEAE-Sepharose, p-hydroxyacetophenone, guanidine HCl, and ethylene glycol were all purchased from Sigma. ACES, MgCl2, and propionaldehyde were purchased from Aldrich. Grade I NAD+ was from Roche Applied Science. The Bio-Gel P-6 DG and Bio-Rad Protein Assay reagent were obtained from Bio-Rad. Polyethylene glycol 6000 was from Hampton Research (Laguna Niguel, CA), and the 24-well sitting drop trays were from Charles Supper Co., Inc. (Natick, MA). Protein Preparation and Crystallization—Human ALDH2 E487K cDNA was expressed in E. coli BL21 (DE3) cells using a pT-7-7 expression system previously described (7Farres J. Wang X. Takahashi K. Cunningham S.J. Wang T.T. Weiner H. J. Biol. Chem. 1994; 269: 13854-13860Abstract Full Text PDF PubMed Google Scholar, 16Zheng C.F. Wang T.T. Weiner H. Alcohol. Clin. Exp. Res. 1993; 17: 828-831Crossref PubMed Scopus (67) Google Scholar). The mutant enzyme was isolated by first employing DEAE-Sepharose chromatography as previously published (16Zheng C.F. Wang T.T. Weiner H. Alcohol. Clin. Exp. Res. 1993; 17: 828-831Crossref PubMed Scopus (67) Google Scholar, 17Jeng J.J. Weiner H. Arch. Biochem. Biophys. 1991; 289: 214-222Crossref PubMed Scopus (43) Google Scholar) with a slight modification of the protocol; the pH of the sodium pyrophosphate buffer was changed to 6.6. Fractions containing ALDH2 were pooled and further purified on a p-hydroxyacetophenone affinity column as described previously (18Ghenbot G. Weiner H. Protein Expression Purif. 1992; 3: 470-478Crossref PubMed Scopus (53) Google Scholar). The eluate was dialyzed in 4 liters of 10 mm ACES buffer, 1 mm dithiothreitol, pH 6.6, for 4 h. Eluate was then transferred to fresh dialysis buffer for an additional 4 h. Protein was concentrated in an Amicon stirred cell and then stored at -20 °C in a 50% glycerol solution. Before each crystallization tray preparation, protein was removed from -20 °C, and buffer was exchanged on a P6 gel column into 10 mm ACES buffer, 2 mm dithiothreitol, pH 6.6. Protein was concentrated to 8 mg/ml in a YM30 Amicon Centricon spinning at 2,000 × g. Protein concentration was monitored by the Bio-Rad Protein Assay using bovine serum albumin as the standard. Enzyme activity was assayed at 25 °C in 100 mm sodium pyrophosphate HCl, pH 9.5, containing 10 mm NAD+ and 200 μm propionaldehyde. NADH production was monitored at 340 nm with a Beckman DU 640 spectrophotometer. For crystal growth, sitting drop trays were set up at 20 °C in a glove box under nitrogen while maintaining a gaseous oxygen concentration less than 2%. Crystals were grown using the sitting drop configuration for vapor diffusion in solutions containing 100 mm ACES, pH 6.2-6.8, 10 mm MgCl2, 100 mm guanidine HCl, 15-17% (w/v) polyethylene glycol 6000, and 4-6 mm dithiothreitol. The equilibrium drop volume was 3 μl. Trays were stored at 20 °C, and crystals were harvested at 3-4 weeks. Flash freezing of the crystals was accomplished using a rapid two-step procedure to introduce 22% ethylene glycol cryoprotectant. The crystals were immediately flash frozen at 110 K in a N2 cryostream. Data Collection, Processing, and Model Refinement—Data were collected at the Advanced Photon Source at Argonne National Laboratory (Argonne, IL) on beamline SBC-CAT 19ID. The beamline was equipped with the APS-1 3 × 3 array CCD detector. The x-ray beam wavelength for data collection was 12.4 keV. Data were indexed, integrated, and scaled with the HKL2000 program suite (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Phases were solved using the molecular replacement method with the human ALDH2 coordinates as a starting model. Molecular replacement was executed using the program AMoRe as implemented in the CCP4 program package (20Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar, 21Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar). The resulting models were refined using the Crystallography and NMR System (CNS) suite (22Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). For the refinement of the initial apoenzyme model, rigid body refinement was followed by simulated annealing and minimization that were performed with NCS restraints applied at a weight of 100 kcal/mol·Å2 for the main-chain atoms and 10 kcal/mol·Å2 for the side-chain atoms. This was followed by individual restrained isotropic temperature factor refinement. Model rebuilding was performed using the program O (23Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). To reduce model bias, composite annealed omit maps were calculated. Only energy minimization and temperature factor refinements were utilized in subsequent rounds with residues 246-264 and 466-476 excluded from NCS restraints during minimization. In the final round of refinement, the NCS restraint weights were reduced to 50 kcal/mol·Å2 for main-chain atoms with no NCS restraints imposed on the side-chain atoms. In this final round, residues 244-271, 424-425, and 463-478 were excluded from the main-chain NCS restraints. Rfree was evaluated using a randomly chosen 5% of the crystallographic data. Structure Analysis—Hinge axis determination for positional domain differences between ALDH2 and ALDH2*2 were analyzed using the program DynDom as implemented in the CCP4 package (20Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar, 24Hayward S. Berendsen H.J.C. Proteins. 1998; 30: 144-154Crossref PubMed Scopus (712) Google Scholar). Tetramer, subunit, and domain alignments were performed using the program LSQKAB as implemented in the CCP4 package (20Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar, 25Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2380) Google Scholar). Root mean square deviations were calculated for the Cα atoms of each alignment. Residues 244-271, 424-425, and 463-478 were omitted for all pairings. To determine the extent of domain reorientation relative to wild type, the cofactor-binding domain was established as the point of reference. This domain was aligned to apoenzyme wild type. The rotated ALDH2*2 subunit was then realigned to wild type using either the catalytic or oligomerization domain. The extent of domain rotation is thus expressed as the amount of rotation necessary to align the domain of interest relative to the common positions of the coenzyme-binding domains. The extent of secondary structural shifts was determined by aligning the cofactor-binding and catalytic domains together to wild-type enzyme. Therefore, these values are expressed as the amount of shift within the context of these domains and are determined independently of domain shifts. Residues were considered ordered and were included in the final coordinates if the electron density maps contoured at one S.D. of the map indicated their presence. ALDH2*2 crystallized in the triclinic space group with three tetramers in the asymmetric unit. The crystal diffracted to 2.1 Å resolution, and the structure was refined to Rfree = 23.8% and Rwork = 20.4%. Like wild-type ALDH2, ALDH2*2 is a tetramer of four identical subunits. Ordered solvent molecules included 2649 water molecules, 21 ethylene glycol molecules, 8 guanidines, and 12 sodium ions. All data collection and refinement statistics are summarized in Table I.Table IData collection and refinement statisticsApo-ALDH2*2Resolution (Å)50 to 2.10Total/unique observations668,406/345,164Completeness (%)aValues for highest resolution shell are in parentheses.97.7 (96.2)〈I/σ(i)〉14.6 (2.3)Rmerge (%)5.1 (35.3)Rwork (%)20.4Rfree (%)bRfree was determined from a randomly selected 5% of the reflections.23.8Space groupP1Cell dimensionsa, b, c (Å)96, 105, 163α, β, γ (degrees)79, 82, 88Asymmetric unit3 tetramersRoot mean square deviation bonds (Å)0.009Root mean square deviation angles (degrees)1.36NCS root mean square deviationcFor Cα atoms; excludes residues 244-271, 424-425, and 463-478. (Å)0.136Overall B-factor (Å2)40.9No. of solvent molecules2690Protein Data Bank ID code1ZUMa Values for highest resolution shell are in parentheses.b Rfree was determined from a randomly selected 5% of the reflections.c For Cα atoms; excludes residues 244-271, 424-425, and 463-478. Open table in a new tab Interactions Surrounding Lys487—The majority of residue Lys487 is well ordered and clearly present in the electron density maps contoured at one S.D. (Fig. 3a). The loss of hydrogen bonds among residues 487, 475, and 264 is accompanied by changes in side-chain conformation of these residues (Fig. 4). No favorable interactions were observed between Lys487 and Arg475 in the dimer partner. Lys487 points away from the dimer interface and forms no new hydrogen bonds. Lys487 does make van der Waals contacts with the side chain of Tyr468 (when observed) in the neighboring subunit as well as with the peptide carbonyl oxygen of Pro158 and the main chain and side chain of Val159 within its own subunit. Arg475 can only be modeled in seven of the 12 subunits. In each of the seven instances, residue 475 occupies the same conformation as wild type with the Nϵ atom within hydrogen bonding distance of the main-chain carbonyl of 488 in the neighboring subunit. The five remaining subunits do not display interpretable electron density for Arg475. Although not identical, all observed conformations for Arg264 are rotated away from Lys487.Fig. 4A stereo diagram of side-chain and main-chain shifts from the dimer interface to the NAD+-binding cleft. NAD+-bound wild type (violet, subunit A; blue, subunit B) and apoenzyme ALDH2*2 (red) are aligned.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Disorder at the Interface—The most striking feature of the ALDH2*2 crystal structure is the disordered region surrounding Lys487 including residues 244-272 and 463-478 (Table II). Adjacent to 469 in the catalytic domain, residues 424 and 425 are also disordered. Residues 469-473 are disordered in all subunits of the asymmetric unit. In 11 of the 12 subunits within the asymmetric unit, residues 248-262 are not visible in electron density maps contoured at one S.D. (Fig. 3b). These residues comprise the αG helix located at the interface between adjacent subunits within a dimer pair. This helix also comprises part of the adenine binding pocket for NAD+. In subunit A, αG is more ordered with only residues 257-263 missing. Although this helix is observed, it has shifted 3 Å, relative to the wild-type structure (Fig. 2b). In the remaining subunits, the general path of αG appears to shift in the same manner as in subunit A; however, the residues are insufficiently defined in the electron density maps for confident placement.Table IIDisordered residuesSubunitαG helixLoopLoopA257-263424-425463-478B244-264424-425468-473C246-262424-425468-474D248-263424-425469-476E246-262424-425469-474F246-262424-425468-474G246-262424-425469-474H246-262424-425469-474I246-263424-425469-474J246-263424-425469-475K246-271424-425466-475L245-263424-425466-476 Open table in a new tab Unique to subunit A are the crystal packing contacts between this subunit and the crystallographically related subunit K. The αJ helix of subunit K makes contact with and stabilizes αF of subunit A. This results in a 15% lower average temperature factor for αF as compared with the rest of the subunit. αF is positioned on the opposite side of the NAD+-binding cleft from αG, and its increased order supports αG in this subunit. Adjacent to αG in both primary and tertiary structure is the β11 strand, which includes Arg264 and Glu268. In all subunits of ALDH2*2, the position of the β11 strand is shifted 2 Å in the same direction as αG. The Cα atom of the putative general base, Glu268, has shifted ∼1.5 Å away from the active site, and its side-chain position is ill defined and not consistent among the subunits. In response to the shifts of both αG and β11, β10 has shifted 0.8 Å in the same direction as αG and β11. Loop Disorder Linked to Active Site—In wild-type ALDH2, the loop composed of residues 463-478 interacts with residues 269-272 through two hydrogen bonds: one between the peptide carbonyl oxygen of 472 and the main-chain nitrogen of 269 and the other between the side-chain hydroxyl of Ser471 and the peptide carbonyl oxygen of residue 270 (Fig. 4). Residues 269-272 associate with the active site through a hydrogen bond between the peptide nitrogen of 271 and the side chain of Glu399. In cofactor-bound wild-type ALDH2, Glu399 stabilizes the nicotinamide portion of NAD+ by forming hydrogen bonds between its carboxylate and the 2′- and 3′-hydroxyl oxygens of the nicotinamide ribose (hydride transfer conformation). However, in concert with the flexibility of the loop containing residues 463-478 in ALDH2*2, the main chain of residues 269-272 rotates such that the amino nitrogen of 271 no longer stabilizes the side chain of Glu399 in the active site (Fig. 4). Glu399 assumes a new conformation that is rotated away from the active site and is instead stabilized by the peptide nitrogen of 272. Coincident with this change, the side chain of Phe401 rotates into the void created by the movement of Glu399. Phe401 supports the binding of the NMN portion of the coenzyme in all ALDH2 holoenzyme structures. The Active Site—The catalytic nucleophile, cysteine 302, is well ordered and retains a wild-type-like conformation. Electron density maps surrounding Cys302 reveal binding of an unknown molecule. Kinetics experiments have shown that the crystallization precipitant, polyethylene glycol 6000MW, contains aldehydes at an approximate concentration of 0.5 mm (12Perez-Miller S. Hurley T.D. Biochemistry. 2003; 42: 7100-7109Crossref PubMed Scopus (142) Google Scholar). Therefore, this unaccounted for density is probably due to unidentified aldehydes arising from the polyethylene glycol. The resolution of the data is insufficient to fully define the bound molecule; therefore, solvent molecules were modeled into the positive difference peaks to partially account for the unknown molecule. Asparigine 169, important for stabilizing oxyanion formation in the transition state (8Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure. 1997; 5: 701-711Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), is also well ordered and is in the same conformation as wild-type enzyme. As mentioned above, the side chain of Glu268 is disordered and not consistent among subunits. Domain Shifts—Between the cofactor-binding domain and the oligomerization domain lies a hinge axis about which there is a 2.5° rotation in ALDH2*2 relative to wild type, as determined using the program DynDom (20Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar, 24Hayward S. Berendsen H.J.C. Proteins. 1998; 30: 144-154Crossref PubMed Scopus (712) Google Scholar). Analysis of relative domain positions within the dimer pair revealed residues that form the hinge, which include the three β strands of the oligomerization domain as well as the loop composed of residues 463-478. Individual domain alignments using the tetramer, dimer, and monomer were also performed (Table III). These, too, demonstrate that the cofactor-binding and catalytic domains have changed position within the tetramer in a manner that can best be described as a 2.5° rigid body rotation away from the dimer interfaces (Fig. 5).Table IIIRoot mean square deviations of Cα atoms for alignments of ALDH2*2 to apoenzyme wild-type ALDH2 (Å2)DomainAll domainsOligomerizationCoenzyme/catalyticCoenzymeCatalyticWild-type tet1 to 2*2 tet10.750.410.770.780.71Wild-type ab to 2*2 ab0.740.390.760.670.68Wild-type a to 2*2 a0.300.340.220.190.222*2 a to 2*2 b0.170.150.140.130.10 Open table in a new tab The crystal structure studies of ALDH2*2 have revealed that mutation of glutamate to lysine at residue 487 causes a large structural transition within the variant enzyme. Whereas Lys487 is not itself disordered in the ALDH2*2 crystal structure, its presence at the dimer interface perturbs local secondary structure elements, particularly αG, β10, β11, and the loop consisting of residues 463-478. These structures are either closely or directly involved with NAD+ binding. The disruptions of αG and the aforementioned loop also lead to an unstable dimer interface where the catalytic and coenzyme-binding domains of individual subunits rotate away from the interface. Moreover, the general base, Glu268, is not stably positioned for catalysis, and the active site is further disrupted at residues 399 and 401 through a cascade of changes that appear to originate in the dimer partner. These shifted main-chain and side-chain positions undoubtedly contribute to the decreased activity observed for this ALDH2 variant and also shed light on the variant's mechanism of allelic dominance. Disorder of residues 463-478 has been observed in other aldehyde dehydrogenase crystal structures. Lamb and Newcomer have reported these residues missing in the electron density maps of cofactor-bound retinal dehydrogenase type II (RalDH2), which shares about 67% sequence identity with ALDH2 (26Lamb A.L. Newcomer M.E. Biochemistry. 19" @default.
• W1969727059 created "2016-06-24" @default.
• W1969727059 creator A5012958448 @default.
• W1969727059 creator A5031403355 @default.
• W1969727059 creator A5046722187 @default.
• W1969727059 date "2005-08-01" @default.
• W1969727059 modified "2023-10-01" @default.
• W1969727059 title "Disruption of the Coenzyme Binding Site and Dimer Interface Revealed in the Crystal Structure of Mitochondrial Aldehyde Dehydrogenase “Asian” Variant" @default.
• W1969727059 cites W1484196488 @default.
• W1969727059 cites W1503086187 @default.
• W1969727059 cites W1539796472 @default.
• W1969727059 cites W1965270719 @default.
• W1969727059 cites W1968105649 @default.
• W1969727059 cites W1975480828 @default.
• W1969727059 cites W1981381364 @default.
• W1969727059 cites W1993709190 @default.
• W1969727059 cites W1993758973 @default.
• W1969727059 cites W1995017064 @default.
• W1969727059 cites W1996881777 @default.
• W1969727059 cites W2001641653 @default.
• W1969727059 cites W2002301555 @default.
• W1969727059 cites W2004146008 @default.
• W1969727059 cites W2007775432 @default.
• W1969727059 cites W2013083986 @default.
• W1969727059 cites W2014908638 @default.
• W1969727059 cites W2023078236 @default.
• W1969727059 cites W2029084148 @default.
• W1969727059 cites W2038121498 @default.
• W1969727059 cites W2044850091 @default.
• W1969727059 cites W2044886069 @default.
• W1969727059 cites W2052943329 @default.
• W1969727059 cites W2054383158 @default.
• W1969727059 cites W2080476827 @default.
• W1969727059 cites W2081718721 @default.
• W1969727059 cites W2086629541 @default.
• W1969727059 cites W2090049751 @default.
• W1969727059 cites W2100235479 @default.
• W1969727059 cites W2136567909 @default.
• W1969727059 cites W4230205158 @default.
• W1969727059 doi "https://doi.org/10.1074/jbc.m502345200" @default.
• W1969727059 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1262676" @default.
• W1969727059 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15983043" @default.
• W1969727059 hasPublicationYear "2005" @default.
• W1969727059 type Work @default.
• W1969727059 sameAs 1969727059 @default.
• W1969727059 citedByCount "95" @default.
• W1969727059 countsByYear W19697270592012 @default.
• W1969727059 countsByYear W19697270592013 @default.
• W1969727059 countsByYear W19697270592014 @default.
• W1969727059 countsByYear W19697270592015 @default.
• W1969727059 countsByYear W19697270592016 @default.
• W1969727059 countsByYear W19697270592017 @default.
• W1969727059 countsByYear W19697270592018 @default.
• W1969727059 countsByYear W19697270592019 @default.
• W1969727059 countsByYear W19697270592020 @default.
• W1969727059 countsByYear W19697270592021 @default.
• W1969727059 countsByYear W19697270592022 @default.
• W1969727059 countsByYear W19697270592023 @default.
• W1969727059 crossrefType "journal-article" @default.
• W1969727059 hasAuthorship W1969727059A5012958448 @default.
• W1969727059 hasAuthorship W1969727059A5031403355 @default.
• W1969727059 hasAuthorship W1969727059A5046722187 @default.
• W1969727059 hasBestOaLocation W19697270591 @default.
• W1969727059 hasConcept C107824862 @default.
• W1969727059 hasConcept C178790620 @default.
• W1969727059 hasConcept C181199279 @default.
• W1969727059 hasConcept C185592680 @default.
• W1969727059 hasConcept C197957613 @default.
• W1969727059 hasConcept C25747580 @default.
• W1969727059 hasConcept C2779546866 @default.
• W1969727059 hasConcept C28859421 @default.
• W1969727059 hasConcept C3175096 @default.
• W1969727059 hasConcept C55493867 @default.
• W1969727059 hasConcept C71240020 @default.
• W1969727059 hasConcept C8010536 @default.
• W1969727059 hasConceptScore W1969727059C107824862 @default.
• W1969727059 hasConceptScore W1969727059C178790620 @default.
• W1969727059 hasConceptScore W1969727059C181199279 @default.
• W1969727059 hasConceptScore W1969727059C185592680 @default.
• W1969727059 hasConceptScore W1969727059C197957613 @default.
• W1969727059 hasConceptScore W1969727059C25747580 @default.
• W1969727059 hasConceptScore W1969727059C2779546866 @default.
• W1969727059 hasConceptScore W1969727059C28859421 @default.
• W1969727059 hasConceptScore W1969727059C3175096 @default.
• W1969727059 hasConceptScore W1969727059C55493867 @default.
• W1969727059 hasConceptScore W1969727059C71240020 @default.
• W1969727059 hasConceptScore W1969727059C8010536 @default.
• W1969727059 hasIssue "34" @default.
• W1969727059 hasLocation W19697270591 @default.
• W1969727059 hasLocation W19697270592 @default.
• W1969727059 hasLocation W19697270593 @default.
• W1969727059 hasOpenAccess W1969727059 @default.
• W1969727059 hasPrimaryLocation W19697270591 @default.
• W1969727059 hasRelatedWork W123874179 @default.
• W1969727059 hasRelatedWork W1967657416 @default.
• W1969727059 hasRelatedWork W1969727059 @default.
• W1969727059 hasRelatedWork W2014144048 @default.
• W1969727059 hasRelatedWork W2043747644 @default.

• next