On the Enzymatic Activation of NADH

Atomic (1 Å) resolution x-ray structures of horse liver alcohol dehydrogenase in complex with NADH revealed the formation of an adduct in the active site between a metal-bound water and NADH. Furthermore, a pronounced distortion of the pyridine ring of NADH was observed. A series of quantum chemical calculations on the water-nicotinamide adduct showed that the puckering of the pyridine ring in the crystal structures can only be reproduced when the water is considered a hydroxide ion. These observations provide fundamental insight into the enzymatic activation of NADH for hydride transfer. Atomic (1 Å) resolution x-ray structures of horse liver alcohol dehydrogenase in complex with NADH revealed the formation of an adduct in the active site between a metal-bound water and NADH. Furthermore, a pronounced distortion of the pyridine ring of NADH was observed. A series of quantum chemical calculations on the water-nicotinamide adduct showed that the puckering of the pyridine ring in the crystal structures can only be reproduced when the water is considered a hydroxide ion. These observations provide fundamental insight into the enzymatic activation of NADH for hydride transfer. liver alcohol dehydrogenase 2-methyl 2,4-pentanediol reduced nicotinamide Nicotinamide adenine dinucleotide NAD(H) is the most abundant electron carrier in cell metabolism. It exists in an oxidized (NAD+) and a reduced (NADH) form, and both species are stable under physiological conditions. Its capacity as a redox agent is exploited by numerous enzymes that catalyze reactions in which NAD+ is reduced to NADH and vice versa. The interconversion of NAD+ and NADH is achieved by the transfer of a hydride ion (two electrons and a proton) between a substrate and NAD(H). A study on deuterated model compounds has shown that the hydride carrier is the C-4 atom on the pyridine ring of the nicotinamide and that the transfer process is stereospecific (Ref.1Loewus F.A. Westheimer F.H. Vennesland B. J. Am. Chem. Soc. 1953; 75: 5018-5023Crossref Scopus (69) Google Scholar and Fig. 1). Theoretical calculations on the nicotinamide indicated that deformation of the pyridine ring into a boat conformation enhances hydride transfer (2Almarsson O. Bruice T.C. J. Am. Chem. Soc. 1993; 115: 2125-2138Crossref Scopus (126) Google Scholar). It has been observed by UV-visible spectroscopy and NMR that the puckering of the pyridine ring changes upon enzyme binding (3Burke J.R. Frey P.A. Biochemistry. 1993; 32: 13220-13230Crossref PubMed Scopus (34) Google Scholar). Until now it was unclear how an enzyme could steer the puckering of the ring to facilitate hydride transfer. Here, we present for the first time an example of enzymatic activation of NADH as it was found in a complex of NADH with horse liver alcohol dehydrogenase (LADH; EC.1.1.1.1).1 LADH is an NAD(H)-dependent enzyme that catalyzes the oxidation of primary and secondary alcohols to aldehydes (4Eklund H. Brändén C.I. Jurnak F.A. McPherson A. Biological Macromolecules and Assemblies. John Wiley 167: 195-201Crossref PubMed Scopus (247) Google Scholar), which also contains yeast alcohol dehydrogenase and the human zinc-dependent alcohol dehydrogenase. The yeast enzyme is the key mediator of alcohol production, whereas the human enzyme cleanses the blood from poisonous compounds. Other functions that have been assigned to the human enzyme are the involvement in the hormonal household by switching on and off steroids like testosterone. It has been associated with the production and neutralization of free radicals, thereby protecting the organism against DNA damage (6Mantle D. Preedy V.R. Adverse Drug React. Toxicol. Rev. 1999; 18: 235-252PubMed Google Scholar). Finally, alcohol dehydrogenase is thought to play a role in the development of the organism through retinoid regulation (7Foglio M.H. Duester G. Biochim. Biophys. Acta. 1999; 1432: 239-250Crossref PubMed Scopus (18) Google Scholar). Retinoids are vitamin A derivatives that regulate the expression of various genes involved in embryonic growth. It has been observed that heavy drinking leads to retarded growth and night blindness. This can be explained by the fact that the continuous oxidation of ethanol interferes with the reduction of carotene to vitamin A. LADH is a homodimer with 374 residues and two zinc sites in each monomer. One zinc is thought to play a structural role, and the other forms the core of the active site. The latter is liganded to two cysteine residues (Cys46 and Cys174) and a histidine (His67). The x-ray structure of the apo-enzyme indicated that the fourth metal ligand was a water molecule (4Eklund H. Brändén C.I. Jurnak F.A. McPherson A. Biological Macromolecules and Assemblies. John Wiley 36: 8743-8754Crossref PubMed Scopus (16) Google Scholar), but the actual mechanism for hydride transfer and proton release is still under debate (10Northrup D.B. Cho Y.-K. Biophys. J. 2000; 79: 1621-1628Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Alternative reaction mechanisms have been proposed for LADH. In one of these (4Eklund H. Brändén C.I. Jurnak F.A. McPherson A. Biological Macromolecules and Assemblies. John Wiley 16: 111-116Crossref PubMed Scopus (51) Google Scholar), the water molecule is linked to the zinc ion during the reaction and goes through a cycle of (de)protonation. This mechanism demands a five coordination around the metal. Such coordination of the metal is thought to be highly improbable, because a fifth ligand would cause collisions with surrounding residues (4Eklund H. Brändén C.I. Jurnak F.A. McPherson A. Biological Macromolecules and Assemblies. John Wiley 28: 9944-9949Crossref PubMed Scopus (32) Google Scholar) and NMR (14Sloan D.L. Young J.M. Mildvan A.S. Biochemistry. 1975; 14: 1998-2008Crossref PubMed Scopus (46) Google Scholar) data on a cobalt-substituted enzyme and perturbed angular correlation of γ-rays measurements (15Hemmingsen L. Bauer R. Bjerrum M.J. Zeppezauer M. Adolph H.W. Formicka G. Cedergren-Zeppezauer E.S. Biochemistry. 1995; 34: 7145-7153Crossref PubMed Scopus (35) Google Scholar) on cadmium-substituted enzyme indicate the existence of a five coordinate intermediate. Unbound NADH displays an absorption maximum at 340 nm in the UV-visible range. When NADH is bound to LADH, the maximum shifts to 325 nm. Early on, it was proposed that this blue shift is caused by the formation of a bond between the catalytic zinc ion and the nicotinamide (16Mahler H.R. Douglas J.J. J. Am. Chem. Soc. 1957; 79: 1159-1166Crossref Scopus (41) Google Scholar). This proposal was discarded after elucidation of the 3 Å resolution x-ray structure of the NADH-LADH complex because it was concluded that the distance between the zinc ion and the nicotinamide was too long. To access the minute structural changes that an enzymatic environment might impose on NAD(H), it is necessary to collect structural data at a truly atomic level. Atomic resolution x-ray data provide an accuracy in atomic positions in the range of 0.03 Å (17Dauter Z. Lamzin V.S. Wilson K.S. Curr. Opin. Struct. Biol. 1997; 7: 681-688Crossref PubMed Scopus (122) Google Scholar, 18Kachalova G.S. Popov A.N. Bartunik H.D. Science. 1999; 284: 473-476Crossref PubMed Scopus (304) Google Scholar), and detailed features of the active site of the protein structure become visible that are lost at lower resolution. Once the precise positions of all the atoms that are essential for the reaction to proceed are known, detailed theoretical studies can be launched to unravel the reaction mechanism. EE-isozyme of LADH was prepared according to procedures described in Ref. 19Hubatsch I. Maurer P. Engel D. Adolph H.W. J. Chromatogr. 1995; 711: 105-112Crossref Scopus (6) Google Scholar. Data were collected on native enzyme in complex with 2-methyl 2,4-pentanediol (MPD) and NADH further referred to as the Zn-MPD-NADH-LADH complex. The zinc was replaced by cadmium following protocols described in Refs. 13Maret W. Biochemistry. 1989; 28: 9944-9949Crossref PubMed Scopus (32) Google Scholar and 20Maret W. Andersson I. Dietrich H. Schneider-Bernlöhr H. Einarsson R. Zeppezauer M. Eur. J. Biochem. 1979; 98: 501-512Crossref PubMed Scopus (123) Google Scholar. A cadmium-substituted enzyme was complexed with MPD and NADH and is further referred to as the Cd-MPD-NADH-LADH complex. The Zn-MPD-NADH-LADH complex was crystallized with polyethylene glycol 400 at a concentration of 20% in 0.05m Tris-HCl buffer at pH 8.2 with 0.5% MPD present. The Cd-MPD-NADH-LADH complex was crystallized by dialysis against 50 mm Tris-HCl, pH 8.2, and stepwise addition of MPD up to a concentration of 12%. Subsequently, polyethylene glycol 400 was added as a cryoprotectant to a final concentration of 20%. Diffraction data were collected on the BW7B beamline at the EMBL Outstation Hamburg, DESY, from flash-cooled crystals, using an image plate from MAR X-ray Research GmbH (Hamburg, Germany). An additional data set was collected on the Cd-MPD-NADH-LADH complex at 277 K. Data were processed, merged, and scaled with the HKL suite (21Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38446) Google Scholar). Statistics are summarized in Table I.Table IData collection and refinement statistics for LADH complexesZn-MPD-NADHCd-MPD-NADHCd-MPD-NADHTemperature (K)120120277Resolution (Å)20 to 1.1020 to 1.1515 to 1.95Measured reflections684,025637,69394,521Unique reflections267,243254,81745,721Space groupP1P1P21Unit cell a, b, c (Å)50.6, 44.1, 93.651.2, 44.6, 94.143.9, 179.0, 50.5α, β, γ (°)104.2, 101.2, 70.9104.3, 101.1, 70.790.0, 107.5, 90.0Overall completeness (%)89.594.586.6OverallI/ς13.119.519.0I/ς highest resolution shell2.01.94.1Rmerge(%) 1-aRmerge = ∑hkl ∑l ‖Ii − 〈I〉‖/∑hkl ∑l 〈I〉, where Ii is an intensity for theith measurement of a reflection with indices hkland 〈I〉 is the weighted mean of the reflection intensity.5.44.23.7Rfactor(%)1-bRfactor = ∑hkl ‖Fo (hkl)‖ − ‖Fc (hkl)∥/∑hkl ‖Fo (hkl)‖, where Fo and Fc are the observed and calculated structure factors, respectively.12.511.916.7Rfree(%)1-cRfree is the crystallographic Rfactor calculated with 0.4% of the data that were excluded from the structure refinement.15.914.521.4Root mean square deviation bonded distances (Å) (Target value: 0.020)0.0160.0150.014Root mean square deviation angle bonded distances (Å) (Target value: 0.040)0.0350.0340.040e.s.u. (Å) 1-dAs estimated from Cruickshank's diffraction precision indicator (33).0.030.030.201-a Rmerge = ∑hkl ∑l ‖Ii − 〈I〉‖/∑hkl ∑l 〈I〉, where Ii is an intensity for theith measurement of a reflection with indices hkland 〈I〉 is the weighted mean of the reflection intensity.1-b Rfactor = ∑hkl ‖Fo (hkl)‖ − ‖Fc (hkl)∥/∑hkl ‖Fo (hkl)‖, where Fo and Fc are the observed and calculated structure factors, respectively.1-c Rfree is the crystallographic Rfactor calculated with 0.4% of the data that were excluded from the structure refinement.1-d As estimated from Cruickshank's diffraction precision indicator (33Cruickshank D.W.J.,. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 583-601Crossref PubMed Scopus (501) Google Scholar). Open table in a new tab Rfree was used to cross-validate the refinement protocol. The “free” reflections were included in the final refinement round. The Zn-Me2SO-NADH-LADH structure (Protein Data Bank reference code 2OHX; Ref. 22Al-Karadaghi S. Cedergren-Zeppezauer E.S. Hovmöller S. Petratos K. Dauter Z. Wilson K.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 793-807Crossref PubMed Scopus (85) Google Scholar) determined to a resolution of 1.8 Å was used as an initial model for refinement. The 1 Å resolution structures were refined with SHELX-97 (23Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 276: 319-343Crossref Scopus (1877) Google Scholar) and REFMAC (24Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1007) Google Scholar) using anisotropic displacement parameters and H atoms at idealized positions. The resolution of the x-ray data allowed the geometry of the nicotinamide of NADH to be freed from restraints to get an accurate picture of the puckering of the pyridine ring. No restraints were applied either to metal-ligand distances. The Cd-MPD-LADH-NADH complex collected at 277 K was refined using the cryo structure as a starting model. Because of the low number of observations, it was not possible to release the restraints in the active site in this case. X-ray structure factor amplitudes and the derived atoms coordinates have been deposited in the Protein Data Bank under accession numbers R1HEUSF,1HEU, R1HF3SF, 1HF3, R1HETSF, and 1HET. Single crystal polarized absorption spectra were recorded at 283 K using a Zeiss MpM800 microspectrophotometer on a crystal placed in a quartz flow cell with the incident beam perpendicular to the face of the crystal (25Merli A. Brodersen D.E. Morini B. Chen Z. Durley R.C.E. Matthews F.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The crystal was with either of the two to the of the polarized The nicotinamide was in a that the the to the was replaced by a coordinates for this nicotinamide were from the Zn-MPD-NADH-LADH crystal and the geometry was at the using the S. M. J. Chem. 1993; 14: Scopus Google Scholar) with a The that the geometry of the nicotinamide were by the and 1 A water molecule was placed at Å from of the pyridine ring of the The geometry of the nicotinamide was to the new with applied to the of the water molecule to the water in a to the pyridine ring as observed in the Zn-MPD-NADH-LADH x-ray the reduced nicotinamide these calculations were to further the environment of the nicotinamide by the of placed at all positions. Fig. 1 the enzyme environment in the crystal structure and the used for the and were at positions. Finally, the zinc atom was included in a with the two and an cysteine and histidine in the crystal X-ray data were collected to a resolution of 1 Å on native zinc LADH and the cadmium-substituted enzyme in complex with NADH and MPD MPD was used as a agent during of the cadmium-substituted protein but only as a addition in of the zinc MPD an complex in both were under conditions. A data set was collected on the Cd-MPD-NADH-LADH complex at 277 K to a resolution of Å that has shown that were no structural changes in the active site by In both of the Zn-MPD-NADH-LADH complex is a to the atom of the nicotinamide in a and This is coordination distance of the catalytic zinc ion. of and distance from the metal is also in both The second to the by a atom of a substrate (4Eklund H. Brändén C.I. Jurnak F.A. McPherson A. Biological Macromolecules and Assemblies. John Wiley 16: 111-116Crossref PubMed Scopus (51) Google Scholar). It was that a five coordination of the metal leads to collisions with surrounding residues with the NAD(H) (4Eklund H. Brändén C.I. Jurnak F.A. McPherson A. Biological Macromolecules and Assemblies. John Wiley 36: PubMed Scopus Google Scholar), a water is at Å of of the pyridine ring. In a complex of with and Z. A. G. J. P.A. Struct. Biol. 1999; PubMed Scopus Google Scholar), a between a protein and the Å) of the pyridine ring is The distances are from x-ray structures with a which that all the atoms that are to be are at a distance that is in with a of resolution of the x-ray the release of restraints might other complex structures with between the pyridine ring and The an essential of the the enzymatic activation of the The of truly atomic resolution x-ray and quantum chemical calculations the to study enzymatic in and to that NADH is linked to the catalytic metal ion. This is of the of the metal and the of the that the by the enzyme the NAD(H) molecule to become involved in hydride transfer. atomic resolution x-ray studies and theoretical calculations on other to a of hydride transfer. Z. Dauter for in data collection and and at for the and for