The first crystal structure of L-threonine dehydrogenase

L-threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.     

Crystal structure of a ternary complex of the alcohol dehydrogenase from Sulfolobus solfataricus

The crystal structure of a ternary complex of the alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) has been determined at 2.3 A. The asymmetric unit contains a dimer with a NADH and a 2-ethoxyethanol molecule bound to each subunit. The comparison with the apo structure of the enzyme reveals that this medium chain ADH undergoes a substantial conformational change in the apo-holo transition, accompanied by loop movements at the domain interface. The extent of domain closure is similar to that observed for the classical horse liver ADH, although some differences are found which can be related to the different oligomeric states of the enzymes. Compared to its apo form, the SsADH ternary complex shows a change in the ligation state of the active site zinc ion which is no longer bound to Glu69, providing additional evidence of the dynamic role played by the conserved glutamate residue in ADHs. In addition, the structure presented here allows the identification of the substrate site and hence of the residues that are important in the binding of both the substrate and the coenzyme.     

Higher metal-ligand coordination in the catalytic site of cobalt-substituted Thermoanaerobacter brockii alcohol dehydrogenase lowers the barrier for enzyme catalysis

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a zinc-dependent NADP(+)/H-linked class enzyme that reversibly catalyzes the oxidation of secondary alcohols to their corresponding ketones. Cobalt substitution studies of other members of the alcohol dehydrogenase (ADH) family showed that the cobalt-containing ADHs have a similar active site structure but slightly decreased activity compared to wild-type zinc ADHs. In contrast, the cobalt-substituted TbADH (Co-TbADH) exhibits an increase in specific activity compared to the native enzyme [Bogin, O., Peretz, M., and Burstein, Y. (1997) Protein Sci. 6, 450-458]. However, the structural basis underlying this behavior is not yet clear. To shed more light on this issue, we studied the local structure and electronics at the catalytic metal site in Co-TbADH by combining X-ray absorption (XAS) and quantum chemical calculations. Importantly, we show that the first metal-ligand coordination shell of Co-TbADH is distorted compared to its native tetrahedral coordination shell and forms an octahedral structure. This is mediated presumably by the addition of two water molecules and results in more positively charged catalytic metal ions. Recently, we have shown that the metal-ligand coordination number of the zinc ion in TbADH changes dynamically during substrate turnover. These structural changes are associated with a higher coordination number of the native catalytic zinc ion and the consequent buildup of a positive charge. Here we propose that the accumulation of a higher coordination number and positive charge at the catalytic metal ion in TbADH stabilizes the structure of the catalytic transition state and hence lowers the barrier for enzyme catalysis.     

The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover.     

Crystal structure of sorbitol dehydrogenase

Sorbitol dehydrogenase (SDH) is a distant relative to the alcohol dehydrogenases (ADHs) with sequence identities around 20%. SDH is a tetramer with one zinc ion per subunit. We have crystallized rat SDH and determined the structure by molecular replacement using a tetrameric bacterial ADH as search object. The conformation of the bound coenzyme is extended and similar to NADH bound to mammalian ADH but the interactions with the NMN-part have several differences with those of ADH. The active site zinc coordination in SDH is significantly different than in mammalian ADH but similar to the one found in the bacterial tetrameric NADP(H)-dependent ADH of Clostridiim beijerinckii. The substrate cleft is significantly more polar than for mammalian ADH and a number of residues are ideally located to position the sorbitol molecule in the active site. The SDH molecule can be considered to be a dimer of dimers, with subunits A-B and C-D, where the dimer interactions are similar to those in mammalian ADH. The tetramers are composed of two of these dimers, which interact with their surfaces opposite the active site clefts, which are accessible on the opposite side. In contrast to the dimer interactions, the tetramer-forming interactions are small with only few hydrogen bonds between side-chains.     

A low-resolution 3D model of the tetrameric alcohol dehydrogenase from Sulfolobus solfataricus

We describe the computation of a model of the thermophilic NAD-dependent homotetrameric alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH). Modeling is based on the knowledge that each monomer contains two Zn ions with catalytic and structural function, respectively. In the database of known structures, proteins with similar functions are either dimers containing two zinc ions per monomer or tetramers with one zinc ion per monomer. In any case, the sequence identity of the target to the possible templates is low. A threading procedure is therefore developed which includes constraints taking into account residue conservation both at the zinc ion binding and at the monomer-monomer interaction sites in the tetrameric unit. The model is consistent with previously reported data. Furthermore, cross-linking experiments are described which support the computed tetrameric model.     

Crystal structure of sorbitol dehydrogenase

Sorbitol dehydrogenase (SDH) is a distant relative to the alcohol dehydrogenases (ADHs) with sequence identities around 20%. SDH is a tetramer with one zinc ion per subunit. We have crystallized rat SDH and determined the structure by molecular replacement using a tetrameric bacterial ADH as search object. The conformation of the bound coenzyme is extended and similar to NADH bound to mammalian ADH but the interactions with the NMN-part have several differences with those of ADH. The active site zinc coordination in SDH is significantly different than in mammalian ADH but similar to the one found in the bacterial tetrameric NADP(H)-dependent ADH of Clostridiim beijerinckii. The substrate cleft is significantly more polar than for mammalian ADH and a number of residues are ideally located to position the sorbitol molecule in the active site. The SDH molecule can be considered to be a dimer of dimers, with subunits A–B and C–D, where the dimer interactions are similar to those in mammalian ADH. The tetramers are composed of two of these dimers, which interact with their surfaces opposite the active site clefts, which are accessible on the opposite side. In contrast to the dimer interactions, the tetramer-forming interactions are small with only few hydrogen bonds between side-chains.     

Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 A resolution

Polyhydric alcohols are widely found in nature and can be accumulated to high concentrations as a protection against a variety of environmental stresses. It is only recently, however, that these molecules have been shown to be active in protection against heat stress, specifically in the use of sorbitol by the silverleaf whitefly, Bemisia argentifolii. We have determined the structure of the enzyme responsible for production of sorbitol in Bemisia argentifolii, NADP(H)-dependent ketose reductase (BaKR), to 2.3 A resolution. The structure was solved by multiwavelength anomalous diffraction (MAD) using the anomalous scattering from two zinc atoms bound in the structure, and was refined to an R factor of 21.9 % (R(free)=25.1 %). BaKR belongs to the medium-chain dehydrogenase family and its structure is the first for the sorbitol dehydrogenase branch of this family. The enzyme is tetrameric, with the monomer having a very similar fold to the alcohol dehydrogenases (ADHs). Although the structure determined is for the apo form, a phosphate ion in the active site marks the likely position for the adenyl phosphate of NADP(H). The catalytic zinc ion is tetrahedrally coordinated to Cys41, His66, Glu67 and a water molecule, in a modification of the zinc site usually found in ADHs. This modified zinc site seems likely to be a conserved feature of the sorbitol dehydrogenase sub-family. Comparisons with other members of the ADH family have also enabled us to model a ternary complex of the enzyme, and suggest how structural differences may influence coenzyme binding and substrate specificity in the reduction of fructose to sorbitol.     

The structure of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix

The structure of the recombinant medium chain alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Aeropyrum pernix has been solved by the multiple anomalous dispersion technique using the signal from the naturally occurring zinc ions. The enzyme is a tetramer with 222 point group symmetry. The ADH monomer is formed from a catalytic and a cofactor-binding domain, with the overall fold similar to previously solved ADH structures. The 1.62 A resolution A.pernix ADH structure is that of the holo form, with the cofactor NADH bound into the cleft between the two domains. The electron density found in the active site has been interpreted to be octanoic acid, which has been shown to be an inhibitor of the enzyme. This inhibitor is positioned with its carbonyl oxygen atom forming the fourth ligand of the catalytic zinc ion. The structural zinc ion of each monomer is present at only partial occupancy and in its absence a disulfide bond is formed. The enhanced thermal stability of the A.pernix ADH is thought to arise primarily from increased ionic and hydrophobic interactions on the subunit interfaces.     

Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn²⁺) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn²⁺ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn²⁺ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.     

Nad+ Dependent Alcohol Dehydrogenase from Sulfolobus Solfataricus: Structural and Functional Features

Alcohol dehydrogenase, EC 1.1.1.1 (ADH), from the thermophilic archaebacterium Sulfolobus solfataricus (SsADH) is a strictly NAD⁺-dependent enzyme with an amino acid sequence related to those of horse liver, yeast and Thermoanaerobium brockii ADHs. This enzyme is remarkably thermophilic and thermostable; protein stability is strictly dependent on the presence of structural zinc in the molecule. For its broad substrate specificity, product stereoselectivity and acceptance of NAD⁺-macromolecular derivatives, SsADH appears suitable for laboratory-scale chemoselective synthesis. Moreover, it represents a useful protein model for studying the structure-function-stability relationships in thermophilic enzymes.     

Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD⁺-cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD⁺ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD⁺ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD⁺, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD⁺ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.     

Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids.

The active-site metal ion and the associated ligand amino acids in the NADP-linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site-directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 28:6549-6555) were verified. To determine the role of the putative active-site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (< 5%). Site-directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non-chelating residue, alanine, abolished not only the metal-binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc-binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.     

Quinohemoprotein alcohol dehydrogenases: structure, function, and physiology

Quino(hemo)protein alcohol dehydrogenases (ADH) that have pyrroloquinoline quinone (PQQ) as the prosthetic group are classified into 3 groups, types I, II, and III. Type I ADH is a simple quinoprotein having PQQ as the only prosthetic group, while type II and type III ADHs are quinohemoprotein having heme c as well as PQQ in the catalytic polypeptide. Type II ADH is a soluble periplasmic enzyme and is widely distributed in Proteobacteria such as Pseudomonas, Ralstonia, Comamonas, etc. In contrast, type III ADH is a membrane-bound enzyme working on the periplasmic surface solely in acetic acid bacteria. It consists of three subunits that comprise a quinohemoprotein catalytic subunit, a triheme cytochrome c subunit, and a third subunit of unknown function. The catalytic subunits of all the quino(hemo)protein ADHs have a common structural motif, a quinoprotein-specific superbarrel domain, where PQQ is deeply embedded in the center. In addition, in the type II and type III ADHs this subunit contains a unique heme c domain. Various type II ADHs each have a unique substrate specificity, accepting a wide variety of alcohols, as is discussed on the basis of recent X-ray crystallographic analyses. Electron transfer within both type II and III ADHs is discussed in terms of the intramolecular reaction from PQQ to heme c and also from heme to heme, and in terms of the intermolecular reaction with azurin and ubiquinone, respectively. Unique physiological functions of both types of quinohemoprotein ADHs are also discussed.     

Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134

FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn(2+) coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn(2+) coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD(+) dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD(+) to NADH that is subsequently used for furfural reduction.     

Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.     

Crystal structure of formaldehyde dehydrogenase from Pseudomonas putida: the structural origin of the tightly bound cofactor in nicotinoprotein dehydrogenases

Formaldehyde dehydrogenase from Pseudomonas putida (PFDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase family. The pyridine nucleotide NAD(H) in PFDH, which is distinct from the coenzyme (as cosubstrate) in typical alcohol dehydrogenases (ADHs), is tightly but not covalently bound to the protein and acts as a cofactor. PFDH can catalyze aldehyde dismutations without an external addition of NAD(H). The structural basis of the tightly bound cofactor of PFDH is unknown. The crystal structure of PFDH has been solved by the multiwavelength anomalous diffraction method using intrinsic zinc ions and has been refined at a 1.65 A resolution. The 170-kDa homotetrameric PFDH molecule shows 222 point group symmetry. Although the secondary structure arrangement and the binding mode of catalytic and structural zinc ions in PFDH are similar to those of typical ADHs, a number of loop structures that differ between PFDH and ADHs in their lengths and conformations are observed. A comparison of the present structure of PFDH with that of horse liver ADH, a typical example of an ADH, reveals that a long insertion loop of PFDH shields the adenine part of the bound NAD(+) molecule from the solvent, and a tight hydrogen bond network exists between the insertion loop and the adenine part of the cofactor, which is unique to PFDH. This insertion loop is conserved completely among the aldehyde-dismutating formaldehyde dehydrogenases, whereas it is replaced by a short turn among typical ADHs. Thus, the insertion loop specifically found among the aldehyde-dismutating formaldehyde dehydrogenases is responsible for the tight cofactor binding of these enzymes and explains why PFDH can effectively catalyze alternate oxidation and reduction of aldehydes without the release of cofactor molecule from the enzyme.     

Spectroscopic studies of inhibited alcohol dehydrogenase from Thermoanaerobacter brockii: proposed structure for the catalytic intermediate state

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP(+) as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.     

Cobalt(II)-substituted class III alcohol and sorbitol dehydrogenases from human liver

The catalytic zinc atoms in class III (chi) alcohol dehydrogenase (ADH) and sorbitol dehydrogenase (SDH) from human liver have been specifically removed and replaced by cobalt(II) with a new ultrafiltration technique. The electronic absorption spectrum of class III cobalt ADH (epsiolon 638 = 870 M-1 cm-1) is nearly identical with those of active site substituted horse EE and human class I (beta 1 beta 1) cobalt ADH. Thus, the coordination environment of the catalytic metal is strictly conserved in these enzymes. However, significant differences are noted when the spectra of class III ADH-coenzyme complexes are compared to the corresponding spectra of the horse enzyme. The spectrum of class III ADH.NADH is split into three bands, centered at 680, 638, and 562 nm. The class III ADH.NAD+ species resembles the alkaline form of the corresponding horse enzyme complex but without exhibiting the pH dependence of the latter. These spectral changes underscore the role of the coenzymes in differentially fine tuning the catalytic metal for its particular function in each ADH. The noncatalytic zinc of class III ADH exchanges with cobalt at pH 7.0. While 9 residues out of 15 in the loop surrounding the noncatalytic zinc of class III ADH differ from those of the class I ADH, the electronic absorption spectra of cobalt in the noncatalytic metal site of class III ADH establish that the coordination environment of this site is conserved as well. The spectrum of cobalt SDH differs significantly from those of cobalt ADHs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigating a catalytic mechanism of hyperthermophilic L-threonine dehydrogenase from Pyrococcus horikoshii

Based on our first structural data of L-threonine dehydrogenase (TDH) of Pyrococcus horikoshii (PhTDH), we examined its catalytic mechanism. The structural analysis indicated that a catalytic zinc atom at the active centre of PhTDH is coordinated by four residues (Cys42, His67, Glu68 and Glu152) with low affinity. These residues are highly conserved in alcohol dehydrogenases (ADHs) and TDHs. Several PhTDH mutants were prepared with respect to Glu152 and other residues, relating to the proton relay system that is substantially a rate-limiting step in ADH. It was found that the E152D mutant showed 3-fold higher turnover rate and reduced affinities toward L-threonine and NAD(+), compared to wild-type PhTDH. The kinetic analysis of Glu152 mutants indicated that the carboxyl group of Glu152 is important for expressing the catalytic activity. The results obtained from pH dependency of kinetic parameters suggested that Glu152 to Asp substitution causes the enhancement of deprotonation of His47 or ionization of zinc-bound water and threonine in the enzyme-NAD(+) complex. Furthermore, it was predicted that the access of threonine substrate to the enzyme-NAD(+) complex induces a large conformational change in the active domain of PhTDH. From these results, we propose here that the proton relay system works as a catalytic mechanism of PhTDH.