The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Hans Jörnvall - One of the best experts on this subject based on the ideXlab platform.
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origin and evolution of medium chain Alcohol Dehydrogenases
Chemico-Biological Interactions, 2013Co-Authors: Hans Jörnvall, Bengt Persson, Ella Cederlund, Joel Hedlund, Tomas Bergman, Yvonne KallbergAbstract:Different lines of Alcohol Dehydrogenases (ADHs) have separate superfamily origins, already recognized but now extended and re-evaluated by re-screening of the latest databank update. The short-chain form (SDR) is still the superfamily with most abundant occurrence, most multiple divergence, most prokaryotic emphasis, and most non-complicated architecture. This pattern is compatible with an early appearance at the time of the emergence of prokaryotic cellular life. The medium-chain form (MDR) is also old but second in terms of all the parameters above, and therefore compatible with a second emergence. However, this step appears seemingly earlier than previously considered, and may indicate sub-stages of early emergences at the increased resolution available from the now greater number of data entries. The Zn-MDR origin constitutes a third stage, possibly compatible with the transition to oxidative conditions on earth. Within all these three lines, repeated enzymogeneses gave the present divergence. MDR-ADH origin(s), at a fourth stage, may also be further resolved in multiple or extended modes, but the classical liver MDR-ADH of the liver type can still be traced to a gene duplication ~550 MYA (million years ago), at the early vertebrate radiation, compatible with the post-eon-shift, "Cambrian explosion". Classes and isozymes correspond to subsequent and recent duplicatory events, respectively. They illustrate a peculiar pattern with functional and emerging evolutionary distinctions between parent and emerging lines, suggesting a parallelism between duplicatory and mutational events, now also visible at separate sub-stages. Combined, all forms show distinctive patterns at different levels and illustrate correlations with global events. They further show that simple molecular observations on patterns, multiplicities and occurrence give much information, suggesting common divergence rules not much disturbed by horizontal gene transfers after the initial origins.
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characterization of new medium chain Alcohol Dehydrogenases adds resolution to duplications of the class i iii and the sub class i genes
Chemico-Biological Interactions, 2011Co-Authors: Ella Cederlund, Lars Hjelmqvist, Bengt Persson, Jawed Shafqat, Annika Norin, Joel Hedlund, Andreas P Jonsson, Wingming Keung, Hans JörnvallAbstract:Four additional variants of Alcohol and aldehyde Dehydrogenases have been purified and functionally characterized, and their primary structures have been determined. The results allow conclusions about the structural and evolutionary relationships within the large family of MDR Alcohol Dehydrogenases from characterizations of the pigeon (Columba livia) and dogfish (Scyliorhinus canicula) major liver Alcohol Dehydrogenases. The pigeon enzyme turns out to be of class I type and the dogfish enzyme of class III type. This result gives a third type of evidence, based on purifications and enzyme characterization in lower vertebrates, that the classical liver Alcohol dehydrogenase originated by a gene duplication early in the evolution of vertebrates. It is discernable as the major liver form at about the level in-between cartilaginous and osseous fish. The results also show early divergence within the avian orders. Structures were determined by Edman degradations, making it appropriate to acknowledge the methodological contributions of Pehr Edman during the 65 years since his thesis at Karolinska Institutet, where also the present analyses were performed.
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mycothiol dependent formaldehyde dehydrogenase a prokaryotic medium chain dehydrogenase reductase phylogenetically links different eukaroytic Alcohol Dehydrogenases primary structure conformational modelling and functional correlations
FEBS Journal, 1997Co-Authors: Annika Norin, Bengt Persson, Peter W Van Ophem, Sander R Piersma, Johannis A Duine, Hans JörnvallAbstract:Prokaryotic mycothiol-dependent formaldehyde dehydrogenase has been structurally characterized by peptide analysis of the 360-residue protein chain and by molecular modelling and functional correlation with the conformational properties of zinc-containing Alcohol Dehydrogenases. The structure is found to be a divergent medium-chain dehydrogenase/reductase (MDR), at a phylogenetic position intermediate between the cluster of dimeric Alcohol Dehydrogenases of all classes (including the human forms), and several tetrameric reductases/Dehydrogenases. Molecular modelling and functionally important residues suggest a fold of the mycothiol-dependent formaldehyde dehydrogenase related overall to that of MDR Alcohol Dehydrogenases, with the presence of the catalytic and structural zinc atoms, but otherwise much altered active-site relationships compatible with the different substrate specificity, and an altered loop structure compatible with differences in the quaternary structure. Residues typical of glutathione binding in class-III Alcohol dehydrogenase are not present, consistent with that the mycothiol factor is not closely similar to glutathione. The molecular architecture is different from that of the 'constant' Alcohol Dehydrogenases (of class-III type) and the 'variable' Alcohol Dehydrogenases (of class-I and class-II types), further supporting the unique structure of mycothiol-dependent formaldehyde dehydrogenase. Borders of internal chain-length differences between this and other MDR enzymes coincide in different combinations, supporting the concept of limited changes in loop regions within this whole family of proteins.
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arabidopsis formaldehyde dehydrogenase molecular properties of plant class iii Alcohol dehydrogenase provide further insights into the origins structure and function of plant class p and liver class i Alcohol Dehydrogenases
FEBS Journal, 1996Co-Authors: Carmen M Martinez, Hans Jörnvall, Bengt Persson, Jawed Shafqat, Hakima Achkor, Rosario M Fernandez, Jaume Farres, Xavier ParésAbstract:A glutathione-dependent formaldehyde dehydrogenase (class III Alcohol dehydrogenase) has been characterized from Arabidopsis thulium. This plant enzyme exhibits kinetic and molecular properties in common with the class III forms from mammals, with a Km, for S-hydroxymethylglutathione of 1.4 μM, an anodic electrophoretic mobility (PI: 5.3–5.6) and a cross-reaction with anti-(rat class III Alcohol dehydrogenase) antibodies. The enzyme structure, deduced from the cDNA sequence, fits into the complex system of Alcohol Dehydrogenases and shows that all life forms share the class III protein type. The corresponding mRNA is 1.4 kb and present in all plant organs; a single copy of the gene is found in the genome. The class III structural variability is different from that of the ethanol-active enzyme types in both vertebrates (class I) and plants (class P), although class P conserves more of the class III properties than class I does. Also the enzymatic properties differ between the two ethanol-active classes. Active-site variability and exchanges at essential residues (Leu/Gly57, Asp/Arg115) may explain the distinct kinetics. These patterns are consistent with two different metabolic roles for the ethanol-active enzymes, a more constant function, reduction of acetaldehyde during hypoxia, for class P, and a more variable function, the detoxication of Alcohols and participation in metabolic conversions, for class I. A sequence motif, Pro-Xaa-Ile/Val-Xaa-Gly-His-Glu-Xaa-Xaa-Gly, common to all medium-chain Alcohol Dehydrogenases is defined.
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The Alcohol Dehydrogenase System
Advances in experimental medicine and biology, 1995Co-Authors: Hans Jörnvall, Olle Danielsson, Lars Hjelmqvist, Bengt Persson, Jawed ShafqatAbstract:Alcohol Dehydrogenases of different types are common enzymes in nature. Two of these families, the medium-chain dehydrogenase/reductase family, MDR, and the shortchain dehydrogenase/reductase family, SDR, are well studied and known since long, but have experienced a recent “explosion” of new knowledge, extension and importance. The MDR family includes the classical zinc-containing liver Alcohol Dehydrogenases encompassing the classes of human liver Alcohol dehydrogenase, while the SDR family includes the Drosophila Alcohol dehydrogenase, which has shorter subunits, no similar metal requirements, other sub-domain arrangements with different structural relationships, and other subunit interactions.
Vicente Gotor - One of the best experts on this subject based on the ideXlab platform.
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expanding the scope of Alcohol Dehydrogenases towards bulkier substrates stereo and enantiopreference for α α dihalogenated ketones
Chemcatchem, 2014Co-Authors: Kinga Kedziora, Iván Lavandera, Wolfgang Kroutil, Fabricio R Bisogno, Vicente Gotorfernandez, Jose Montejobernardo, Santiago Garciagranda, Vicente GotorAbstract:Alcohol Dehydrogenases (ADHs) were identified as suitable enzymes for the reduction of the corresponding α,α-dihalogenated ketones, obtaining optically pure β,β-dichloro- or β,β-dibromohydrins with excellent conversions and enantiomeric excess. Among the different biocatalysts tested, ADHs from Rhodococcus ruber (ADH-A), Ralstonia sp. (RasADH), Lactobacillus brevis (LBADH), and PR2ADH proved to be the most efficient ones in terms of activity and stereoselectivity. In a further study, two racemic α-substituted ketones, namely α-bromo- α-chloro- and α-chloro-α-fluoroacetophenone were investigated to obtain one of the four possible diastereoisomers through a dynamic kinetic process. In the case of the brominated derivative, only the (1R)-enantiomer was obtained by using ADH-A, although with moderate diastereomeric excess (>99 % ee, 63 % de), whereas the fluorinated ketone exhibited a lower stereoselectivity (up to 45 % de).
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synthesis of enantiopure fluorohydrins using Alcohol Dehydrogenases at high substrate concentrations
ChemInform, 2013Co-Authors: Wioleta Borzecka, Iván Lavandera, Vicente GotorAbstract:It is shown that different Alcohol Dehydrogenases can be used to synthesize both Alcohol antipodes in an enantiopure form by reduction of α-fluoro ketones.
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synthesis of enantiopure fluorohydrins using Alcohol Dehydrogenases at high substrate concentrations
Journal of Organic Chemistry, 2013Co-Authors: Wioleta Borzecka, Iván Lavandera, Vicente GotorAbstract:The use of purified and overexpressed Alcohol Dehydrogenases to synthesize enantiopure fluorinated Alcohols is shown. When the bioreductions were performed with ADH-A from Rhodococcus ruber overexpressed in E. coli, no external cofactor was necessary to obtain the enantiopure (R)-derivatives. Employing Lactobacillus brevis ADH, it was possible to achieve the synthesis of enantiopure (S)-fluorohydrins at a 0.5 M substrate concentration. Furthermore, due to the activated character of these substrates, a huge excess of the hydrogen donor was not necessary.
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access to enantiopure α alkyl β hydroxy esters through dynamic kinetic resolutions employing purified overexpressed Alcohol Dehydrogenases
Advanced Synthesis & Catalysis, 2012Co-Authors: Anibal Cuetos, Iván Lavandera, Wolfgang Kroutil, Fabricio R Bisogno, Ana Riozmartinez, Barbara Grischek, Gonzalo De Gonzalo, Vicente GotorAbstract:α-Alkyl-β-hydroxy esters were obtained via dynamic kinetic resolution (DKR) employing purified or crude E. coli overexpressed Alcohol Dehydrogenases (ADHs). ADH-A from R. ruber, CPADH from C. parapsilosis and TesADH from T. ethanolicus afforded syn-(2R,3S) derivatives with very high selectivities for sterically not impeded ketones (‘small-bulky’ substrates), while ADHs from S. yanoikuyae (SyADH) and Ralstonia sp. (RasADH) could also accept bulkier keto esters (‘bulky-bulky’ substrates). SyADH also provided preferentially syn-(2R,3S) isomers and RasADH showed in some cases good selectivity towards the formation of anti-(2S,3S) derivatives. With anti-Prelog ADHs such as LBADH from L. brevis or LKADH from L. kefir, syn-(2S,3R) Alcohols were obtained with high conversions and diastereomeric excess in some cases, especially with LBADH. Furthermore, due to the thermodynamically favoured reduction of these substrates, it was possible to employ just a minimal excess of 2-propanol to obtain the final products with quantitative conversions.
Bengt Persson - One of the best experts on this subject based on the ideXlab platform.
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origin and evolution of medium chain Alcohol Dehydrogenases
Chemico-Biological Interactions, 2013Co-Authors: Hans Jörnvall, Bengt Persson, Ella Cederlund, Joel Hedlund, Tomas Bergman, Yvonne KallbergAbstract:Different lines of Alcohol Dehydrogenases (ADHs) have separate superfamily origins, already recognized but now extended and re-evaluated by re-screening of the latest databank update. The short-chain form (SDR) is still the superfamily with most abundant occurrence, most multiple divergence, most prokaryotic emphasis, and most non-complicated architecture. This pattern is compatible with an early appearance at the time of the emergence of prokaryotic cellular life. The medium-chain form (MDR) is also old but second in terms of all the parameters above, and therefore compatible with a second emergence. However, this step appears seemingly earlier than previously considered, and may indicate sub-stages of early emergences at the increased resolution available from the now greater number of data entries. The Zn-MDR origin constitutes a third stage, possibly compatible with the transition to oxidative conditions on earth. Within all these three lines, repeated enzymogeneses gave the present divergence. MDR-ADH origin(s), at a fourth stage, may also be further resolved in multiple or extended modes, but the classical liver MDR-ADH of the liver type can still be traced to a gene duplication ~550 MYA (million years ago), at the early vertebrate radiation, compatible with the post-eon-shift, "Cambrian explosion". Classes and isozymes correspond to subsequent and recent duplicatory events, respectively. They illustrate a peculiar pattern with functional and emerging evolutionary distinctions between parent and emerging lines, suggesting a parallelism between duplicatory and mutational events, now also visible at separate sub-stages. Combined, all forms show distinctive patterns at different levels and illustrate correlations with global events. They further show that simple molecular observations on patterns, multiplicities and occurrence give much information, suggesting common divergence rules not much disturbed by horizontal gene transfers after the initial origins.
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characterization of new medium chain Alcohol Dehydrogenases adds resolution to duplications of the class i iii and the sub class i genes
Chemico-Biological Interactions, 2011Co-Authors: Ella Cederlund, Lars Hjelmqvist, Bengt Persson, Jawed Shafqat, Annika Norin, Joel Hedlund, Andreas P Jonsson, Wingming Keung, Hans JörnvallAbstract:Four additional variants of Alcohol and aldehyde Dehydrogenases have been purified and functionally characterized, and their primary structures have been determined. The results allow conclusions about the structural and evolutionary relationships within the large family of MDR Alcohol Dehydrogenases from characterizations of the pigeon (Columba livia) and dogfish (Scyliorhinus canicula) major liver Alcohol Dehydrogenases. The pigeon enzyme turns out to be of class I type and the dogfish enzyme of class III type. This result gives a third type of evidence, based on purifications and enzyme characterization in lower vertebrates, that the classical liver Alcohol dehydrogenase originated by a gene duplication early in the evolution of vertebrates. It is discernable as the major liver form at about the level in-between cartilaginous and osseous fish. The results also show early divergence within the avian orders. Structures were determined by Edman degradations, making it appropriate to acknowledge the methodological contributions of Pehr Edman during the 65 years since his thesis at Karolinska Institutet, where also the present analyses were performed.
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mycothiol dependent formaldehyde dehydrogenase a prokaryotic medium chain dehydrogenase reductase phylogenetically links different eukaroytic Alcohol Dehydrogenases primary structure conformational modelling and functional correlations
FEBS Journal, 1997Co-Authors: Annika Norin, Bengt Persson, Peter W Van Ophem, Sander R Piersma, Johannis A Duine, Hans JörnvallAbstract:Prokaryotic mycothiol-dependent formaldehyde dehydrogenase has been structurally characterized by peptide analysis of the 360-residue protein chain and by molecular modelling and functional correlation with the conformational properties of zinc-containing Alcohol Dehydrogenases. The structure is found to be a divergent medium-chain dehydrogenase/reductase (MDR), at a phylogenetic position intermediate between the cluster of dimeric Alcohol Dehydrogenases of all classes (including the human forms), and several tetrameric reductases/Dehydrogenases. Molecular modelling and functionally important residues suggest a fold of the mycothiol-dependent formaldehyde dehydrogenase related overall to that of MDR Alcohol Dehydrogenases, with the presence of the catalytic and structural zinc atoms, but otherwise much altered active-site relationships compatible with the different substrate specificity, and an altered loop structure compatible with differences in the quaternary structure. Residues typical of glutathione binding in class-III Alcohol dehydrogenase are not present, consistent with that the mycothiol factor is not closely similar to glutathione. The molecular architecture is different from that of the 'constant' Alcohol Dehydrogenases (of class-III type) and the 'variable' Alcohol Dehydrogenases (of class-I and class-II types), further supporting the unique structure of mycothiol-dependent formaldehyde dehydrogenase. Borders of internal chain-length differences between this and other MDR enzymes coincide in different combinations, supporting the concept of limited changes in loop regions within this whole family of proteins.
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arabidopsis formaldehyde dehydrogenase molecular properties of plant class iii Alcohol dehydrogenase provide further insights into the origins structure and function of plant class p and liver class i Alcohol Dehydrogenases
FEBS Journal, 1996Co-Authors: Carmen M Martinez, Hans Jörnvall, Bengt Persson, Jawed Shafqat, Hakima Achkor, Rosario M Fernandez, Jaume Farres, Xavier ParésAbstract:A glutathione-dependent formaldehyde dehydrogenase (class III Alcohol dehydrogenase) has been characterized from Arabidopsis thulium. This plant enzyme exhibits kinetic and molecular properties in common with the class III forms from mammals, with a Km, for S-hydroxymethylglutathione of 1.4 μM, an anodic electrophoretic mobility (PI: 5.3–5.6) and a cross-reaction with anti-(rat class III Alcohol dehydrogenase) antibodies. The enzyme structure, deduced from the cDNA sequence, fits into the complex system of Alcohol Dehydrogenases and shows that all life forms share the class III protein type. The corresponding mRNA is 1.4 kb and present in all plant organs; a single copy of the gene is found in the genome. The class III structural variability is different from that of the ethanol-active enzyme types in both vertebrates (class I) and plants (class P), although class P conserves more of the class III properties than class I does. Also the enzymatic properties differ between the two ethanol-active classes. Active-site variability and exchanges at essential residues (Leu/Gly57, Asp/Arg115) may explain the distinct kinetics. These patterns are consistent with two different metabolic roles for the ethanol-active enzymes, a more constant function, reduction of acetaldehyde during hypoxia, for class P, and a more variable function, the detoxication of Alcohols and participation in metabolic conversions, for class I. A sequence motif, Pro-Xaa-Ile/Val-Xaa-Gly-His-Glu-Xaa-Xaa-Gly, common to all medium-chain Alcohol Dehydrogenases is defined.
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The Alcohol Dehydrogenase System
Advances in experimental medicine and biology, 1995Co-Authors: Hans Jörnvall, Olle Danielsson, Lars Hjelmqvist, Bengt Persson, Jawed ShafqatAbstract:Alcohol Dehydrogenases of different types are common enzymes in nature. Two of these families, the medium-chain dehydrogenase/reductase family, MDR, and the shortchain dehydrogenase/reductase family, SDR, are well studied and known since long, but have experienced a recent “explosion” of new knowledge, extension and importance. The MDR family includes the classical zinc-containing liver Alcohol Dehydrogenases encompassing the classes of human liver Alcohol dehydrogenase, while the SDR family includes the Drosophila Alcohol dehydrogenase, which has shorter subunits, no similar metal requirements, other sub-domain arrangements with different structural relationships, and other subunit interactions.
Kazunobu Matsushita - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and characterization of two inducible nad adh genes encoding nad dependent Alcohol Dehydrogenases from acetobacter pasteurianus sku1108
Journal of Bioscience and Bioengineering, 2011Co-Authors: Uraiwan Masud, Kazunobu Matsushita, Gunjana TheeragoolAbstract:Abstract The cytosolic NAD + -dependent Alcohol Dehydrogenases (NAD + -ADHs) are induced in the quinoprotein ADH-(PQQ-ADH) defective Acetobacter pasteurianus SKU1108 mutant during growth in an ethanol medium. The a dhI and adhII genes, which encode NAD + -ADH I and ADH II, respectively, of this strain have been cloned and characterized. Sequence analyses have revealed that the adhI gene consists of 1029 bp coding for 342 amino acids, which share 99.71% identity with the same protein from A. pasteurianus IFO 3283. Conversely, the adhII gene is composed of 762 bp encoding for a polypeptide of 253 amino acids, which exhibit 99.60% identity with the A. pasteurianus IFO 3283 protein. ADH I is a member of the group I Zn-dependent long-chain ADHs, while the ADH II belongs to the group II short-chain dehydrogenase/reductase NAD + -ADHs. The NAD + - adh gene disruptants exhibited a growth reduction when grown in an ethanol medium. In Escherichia coli , ethanol induced adhI and adhII promoter activities by approximately 1.5 and 2.0 times, respectively, and the promoter activity of the adhII gene exceeded that of the adhI gene by approximately 3.5 times. The possible promoter regions of the adhI and adhII genes are located at approximately 81–105 bp and 74–92 bp, respectively, from their respective ATG start codons. Their repressor regions might be located in proximity to these promoters and may repress gene expression in the wild-type, where the membrane-bound ADH effectively functions.
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Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein Alcohol Dehydrogenases.
Applied microbiology and biotechnology, 2009Co-Authors: Watchara Kanchanarach, Osao Adachi, Gunjana Theeragool, Toshiharu Yakushi, Hirohide Toyama, Kazunobu MatsushitaAbstract:We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol Dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.
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quinohemoprotein Alcohol Dehydrogenases structure function and physiology
Archives of Biochemistry and Biophysics, 2004Co-Authors: Hirohide Toyama, Osao Adachi, Kazunobu Matsushita, Scott F MathewsAbstract: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.
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three distinct quinoprotein Alcohol Dehydrogenases are expressed when pseudomonas putida is grown on different Alcohols
Journal of Bacteriology, 1995Co-Authors: Hirohide Toyama, Kazunobu Matsushita, A Fujii, Emiko Shinagawa, M Ameyama, Osao AdachiAbstract:A bacterial strain that can utilize several kinds of Alcohols as its sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida HK5. Three distinct dye-linked Alcohol Dehydrogenases (ADHs), each of which contained the prosthetic group pyrroloquinoline quinone (PQQ), were formed in the soluble fractions of this strain grown on different Alcohols. ADH I was formed most abundantly in the cells grown on ethanol and was similar to the quinoprotein ADH reported for P. putida (H. Gorisch and M. Rupp, Antonie Leeuwenhoek 56:35-45, 1989) except for its isoelectric point. The other two ADHs, ADH IIB and ADH IIG, were formed separately in the cells grown on 1-butanol and 1,2-propanediol, respectively. Both of these enzymes contained heme c in addition to PQQ and functioned as quinohemoprotein Dehydrogenases. Potassium ferricyanide was an available electron acceptor for ADHs IIB and IIG but not for ADH I. The molecular weights were estimated to be 69,000 for ADH IIB and 72,000 for ADH IIG, and both enzymes were shown to be monomers. Antibodies raised against each of the purified ADHs could distinguish the ADHs from one another. Immunoblot analysis showed that ADH I was detected in cells grown on each Alcohol tested, but ethanol was the most effective inducer. ADH IIB was formed in the cells grown on Alcohols of medium chain length and also on 1,3-butanediol. Induction of ADH IIG was restricted to 1,2-propanediol or glycerol, of which the former Alcohol was more effective. These results from immunoblot analysis correlated well with the substrate specificities of the respective enzymes. Thus, three distinct quinoprotein ADHs were shown to be synthesized by a single bacterium under different growth conditions.
Bryce V. Plapp - One of the best experts on this subject based on the ideXlab platform.
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activity of yeast Alcohol Dehydrogenases on benzyl Alcohols and benzaldehydes characterization of adh1 from saccharomyces carlsbergensis and transition state analysis
Chemico-Biological Interactions, 2009Co-Authors: Suresh Pal, Doohong Park, Bryce V. PlappAbstract:Abstract The substrate specificities of yeast Alcohol Dehydrogenases I and II from Saccharomyces cerevisiae ( Sce ADH1 and Sce ADH2) and Saccharomyces carlsbergensis ( Scb ADH1) were studied. For this work, the gene for the S. carlsbergensis ADH1 was cloned, sequenced and expressed. The amino acid sequence of Scb ADH1 differs at four positions as compared to Sce ADH1, including substitutions of two glutamine residues with glutamic acid residues, and has the same sequence as the commercial yeast enzyme, which apparently is prepared from S. carlsbergensis . The electrophoretic mobilities of Scb ADH1, Sce ADH2 and commercial ADH are similar. The kinetics and specificities of Scb ADH1 and Sce ADH1 acting on branched, long-chain and benzyl Alcohols are very similar, but the catalytic efficiency of Sce ADH2 is about 10–100-fold higher on these substrates. A three-dimensional structure of Sce ADH1 shows that the substrate binding pocket has Met-270, whereas Sce ADH2 has Leu-270, which allows larger substrates to bind. The reduction of a series of p -substituted benzaldehydes catalyzed by Sce ADH2 is significantly enhanced by electron-withdrawing groups, whereas the oxidation of p -substituted aromatic Alcohols may be only slightly affected by the substituents. The substituent effects on catalysis generally reflect the effects on the equilibrium constant for the reaction, where electron-withdrawing substituents favor Alcohol. The results are consistent with a transition state that is electronically similar to the Alcohol, supporting previous results obtained with commercial yeast ADH.
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formamides mimic aldehydes and inhibit liver Alcohol Dehydrogenases and ethanol metabolism
Journal of Biological Chemistry, 2003Co-Authors: Thulasiram H Venkataramaiah, Bryce V. PlappAbstract:Formamides are unreactive analogues of the aldehyde substrates of Alcohol Dehydrogenases and are useful for structure-function studies and for specific inhibition of Alcohol metabolism. They bind to the enzyme-NADH complex and are uncompetitive inhibitors against varied concentrations of Alcohol. Fourteen new branched chain and chiral formamides were prepared and tested as inhibitors of purified Class I liver Alcohol Dehydrogenases: horse (EqADH E), human (HsADH1C*2), and mouse (MmADH1). In general, larger, substituted formamides, such as N-1-ethylheptylformamide, are better inhibitors of HsADH1C*2 and MmADH1 than of EqADH, reflecting a few differences in amino acid residues that change the sizes of the active sites. In contrast, the linear, alkyl (n-propyl and n-butyl) formamides are better inhibitors of EqADH and MmADH1 than of HsADH1C*2, probably because water disrupts van der Waals interactions. These enzymes are also inhibited strongly by sulfoxides and 4-substituted pyrazoles. The structure of EqADH complexed with NADH and (R)-N-1-methylhexylformamide was determined by x-ray crystallography at 1.6 A resolution. The structure resembles the expected Michaelis complex with NADH and aldehyde, and shows for the first time that the reduced nicotinamide ring of NADH is puckered, as predicted for the transition state for hydride transfer. Metabolism of ethanol in mice was inhibited by several formamides. The data were fitted with kinetic simulation to a mechanism that describes the non-linear progress curves and yields estimates of the in vivo inhibition constants and the rate constants for elimination of inhibitors. Some small formamides, such as N-isopropylformamide, may be useful inhibitors in vivo.
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Uncompetitive Inhibitors of Alcohol Dehydrogenases
Advances in experimental medicine and biology, 1999Co-Authors: Bryce V. Plapp, Vijay K. Chadha, Kevin G. Leidal, Heeyeong Cho, Michael Scholze, John F. Schindler, Kristine B. Berst, S. RamaswamyAbstract:Selective inhibitors of Alcohol Dehydrogenases could be useful for prevention of poisoning due to metabolism of Alcohols, such as methanol or ethylene glycol, that lead to toxic products (Jacobsen and McMartin, 1997). Good inhibitors could also be used to study the physiological functions of the various isoenzymes of Alcohol dehydrogenase and for therapeutic intervention after the metabolic roles of the enzymes are established. Although 4-methylpyrazole is a potent inhibitor of some of the liver Alcohol Dehydrogenases, it is not very effective against all of the human isoenzymes, and it is a competitive inhibitor against Alcohol, which makes it less effective when the concentration of substrate Alcohol is increased. Thus, we have been designing, synthesizing, and evaluating potentially specific inhibitors that are uncompetitive against varied concentrations of Alcohols. The research has led to some selective inhibitors of human Alcohol Dehydrogenases and structure- function information about the specificities of the enzymes.
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Crystallographic investigations of Alcohol Dehydrogenases
EXS, 1994Co-Authors: Hans Eklund, Olle Danielsson, Bryce V. Plapp, S. Ramaswamy, Jan-olov Höög, Mustafa El-ahmad, Hans JörnvallAbstract:The structures of horse liver Alcohol dehydrogenase class I in its apoenzyme form and in different ternary complexes have been determined at high resolution. The complex with NAD+ and the substrate analogue pentafluorobenzyl Alcohol gives a detailed picture of the interactions in an enzyme-substrate complex. The Alcohol is bound to the zinc and positioned so that the hydrogen atom can be directly transferred to the C4 atom of the nicotinamide ring. The structure of cod liver Alcohol dehydrogenase with hybrid properties (functionally of class I but structurally overall closer to class III) has been determined by molecular replacement methods to 3 A resolution. Yeast Alcohol dehydrogenase has been crystallized, and native data have been collected to 3 A resolution.
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Catalysis by Yeast Alcohol Dehydrogenase
Advances in experimental medicine and biology, 1991Co-Authors: Bryce V. Plapp, Axel J. Ganzhorn, Robert M. Gould, David W. Green, Tobias Jacobi, Edda Warth, Darla Ann KratzerAbstract:The structure and mechanism of Alcohol Dehydrogenases have been extensively studied (Branden et al., 1975; Klinman, 1981; Pettersson, 1987). The three-dimensional structures of the horse liver enzyme in several ternary complexes have been solved at high resolution (Eklund et al., 1981, 1982). Amino acid sequences for more than 22 NAD+-dependent Alcohol Dehydrogenases from 11 animal, plant and fungal species are known. Comparison of these sequences raises many questions about the structure-function relationships in these enzymes. How do the amino acid residues at the active site participate in catalysis? What is the basis of substrate specificity? What was selected for during the evolution of the different enzymes?
