Identification of Residues Potentially Involved in the Interactions Between Subunits in Yeast Alcohol Dehydrogenases (original) (raw)
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Biochemistry, 2004
The crystal structure of NAD +-dependent alcohol dehydrogenase from Bacillus stearothermophilus strain LLD-R (htADH) was determined using X-ray diffraction data at a resolution of 2.35 Å. The structure of homotetrameric htADH is highly homologous to those of bacterial and archaeal homotetrameric alcohol dehydrogenases (ADHs) and also to the mammalian dimeric ADHs. There is one catalytic zinc atom and one structural zinc atom per enzyme subunit. The enzyme was crystallized as a binary complex lacking the nicotinamide adenine dinucleotide (NAD +) cofactor but including a zinccoordinated substrate analogue trifluoroethanol. The binary complex structure is in an open conformation similar to ADH structures without the bound cofactor. Features important for the thermostability of htADH are suggested by a comparison with a homologous mesophilic enzyme (55% identity), NAD +-dependent alcohol dehydrogenase from Escherichia coli. To gain insight into the conformational change triggered by NAD + binding, amide hydrogen-deuterium exchange of htADH, in the presence and absence of NAD + , was studied by HPLC-coupled electrospray mass spectrometry. When the deuteron incorporation of the protein-derived peptides was analyzed, it was found that 9 of 21 peptides show some decrease in the level of deuteron incorporation upon NAD + binding, and another 4 peptides display slower exchange rates. With one exception (peptide number 8), none of the peptides that are altered by bound NAD + are in contact with the alcohol-substrate-binding pocket. Furthermore, peptides 5 and 8, which are located outside the NAD +-binding pocket, are notable by displaying changes upon NAD + binding. This suggests that the transition from the open to the closed conformation caused by cofactor binding has some longrange effects on the protein structure and dynamics.
Archives of biochemistry and biophysics, 2016
Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxyl...
Structure and function of yeast alcohol dehydrogenase
Journal of the Serbian Chemical Society, 2000
1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer This article has been corrected. Link to the correction 10.2298/JSC0008609E
Oligomeric integrity-the structural key to thermal stability in bacterial alcohol dehydrogenases
Protein Science, 1999
Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)-dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30° in their melting temperatures. The crystal structures of CBADH and TBADH in their holo-enzyme form have been determined at a resolution of 2.05 and 2.5 Å, respectively. Comparison of these two very similar structures (RMS difference in Ca = 0.8 Å) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, “charged-neutral” hydrogen bonds, and prolines as well as improved stability of α-helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.
International Journal of Biological Macromolecules, 2013
In this study, the dissociative thermal inactivation and conformational lock theories are applied for the homodimeric enzyme glucose oxidase (GOD) in order to analyze its structure. For this purpose, the rate of activity reduction of glucose oxidase is studied at various temperatures using b-D-glucose as the substrate by incubation of enzyme at various temperatures in the wide range between 40 and 70°C using UV-Vis spectrophotometry. It was observed that in the two ranges of temperatures, the enzyme has two different forms. In relatively low temperatures, the enzyme is in its dimeric state and has normal activity. In high temperatures, the activity almost disappears and it aggregates. The above achievements are confirmed by dynamic light scattering. The experimental parameter ''n'' as the obvious number of conformational locks at the dimer interface of glucose oxidase is obtained by kinetic data, and the value is near to two. To confirm the above results, the X-ray crystallography structure of the enzyme, GOD (pdb, 1gal), was also studied. The secondary and tertiary structures of the enzyme to track the thermal inactivation were studied by circular dichroism and fluorescence spectroscopy, respectively. We proposed a mechanism model for thermal inactivation of GOD based on the absence of the monomeric form of the enzyme by circular dichroism and fluorescence spectroscopy.
Binding of coenzymes to yeast alcohol dehydrogenase
Journal of the Serbian Chemical Society, 2010
In this work, the binding of coenzymes to yeast alcohol dehydrogenase (EC 1.1.1.1) were investigated. The main criterions were the change in the standard free energies for individual reaction steps, the internal equilibrium constants and the overall changes in the reaction free energies. The calculations were performed for the wild type enzyme at pH 6-9 and for 15 different mutant type enzymes, with single or double point mutations, at pH 7.3. The abundance of theoretical and experimental data enabled the binding of coenzymes to enzyme to be assessed in depth.
Biochemical Genetics, 1998
Three-dimensional structures of sevenshort-chain dehydrogenases/reductases show that theseenzymes share common structural features. Sequencealignment studies of Drosophila alcoholdehydrogenase (DADH), with an unknown 3D-structure, and fourshort-chain dehydrogenases/reductases with known X-raystructures suggest that DADH shares the same structuralfeatures. However, the substrate binding regions, which are located in the C-terminal region of theseenzymes, share little sequence homology, because of thewide variety of substrates used. X-ray structures ofshort-chain dehydrogenases/reductases indicate that conformational changes occur in a loop, inthe C-terminal region, upon substrate binding. Thissubstrate-binding loop is located between a strand anda helix and may contain one or two small helices. Secondary structure predictions and modelingstudies of this substrate-binding loop in DADH predictthat the two helices may also be present in this enzyme.The naturally occurring variants of Drosophila melanogaster alleloenzymes ADH-S and ADH-Fdiffer in a replacement of threonine by lysine atposition 192, which is located at a central position inthe substrate-binding loop. The positive charge oflysine may move significantly on substrate binding,resulting in a direct charge interaction withNAD+ in the enzyme-substrate complex,explaining a very strong influence of pH on the bindingof ADH-S for the NAD+ analogue Cibacron Blue. Thisindicates that the ADH S/F polymorphism has a directinfluence on the catalytic properties of the enzyme.
Journal of Molecular Biology, 2000
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 Ê and 2.1 A Ê 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 o-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inef®cient 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 Ê 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.
Letters in Peptide Science, 1998
Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophileClostridium beijerinckii (CBADH) and the other from the extreme thermophileThermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26°C in thermal stability. To explore the role of linear segments of these similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed inEscherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.