scholarly journals Purification, crystallization and properties of primary alcohol dehydrogenase from a methanol-oxidizing Pseudomonas sp. No 2941.

1977 ◽  
Vol 41 (3) ◽  
pp. 467-475 ◽  
Author(s):  
Kei YAMANAKA ◽  
Keisuke MATSUMOTO
Keyword(s):  
Alcohol Dehydrogenase ◽  
Primary Alcohol ◽  
Pseudomonas Sp

scholarly journals Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme

1996 ◽  
Vol 316 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Douglas S. BURDETTE ◽  
Claire VIEILLE ◽  
J. Gregory ZEIKUS
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Primary Alcohol ◽  
Recombinant Enzyme ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Arrhenius Plots ◽  
Thermoanaerobacter Ethanolicus ◽  
Secondary Alcohol Dehydrogenase

The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (S-ADH) was cloned, sequenced and expressed in Escherichia coli. The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits. The purified recombinant enzyme is optimally active above 90 °C with a half-life of approx. 1.7 h at 90 °C. An NADP(H)-dependent enzyme, the recombinant S-ADH has 1400-fold greater catalytic efficiency in propan-2-ol oxidation than in ethanol oxidation. The enzyme was inactivated by chemical modification with dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis. Zinc was the only metal enhancing S-ADH reactivation after DTNB modification, implicating the involvement of bound zinc in catalysis. Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant S-ADHs were linear from 25 to 90 °C when the enzymes were incubated at 55 °C before assay. Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0 or 25 °C. The observed Arrhenius discontinuity therefore resulted from a temperature-dependent, catalytically significant S-ADH structural change. Hydrophobic cluster analysis comparisons of both mesophilic and thermophilic S-ADH and primary- versus S-ADH amino acid sequences were performed. These comparisons predicted that specific proline residues might contribute to S-ADH thermostability and thermophilicity, and that the catalytic Zn ligands are different in primary-alcohol dehydrogenases (two Cys and a His) and S-ADHs (Cys, His, and Asp).

Regiospecific horse liver alcohol dehydrogenase-catalyzed oxidations of some bishydroxycyclohexanes

1977 ◽  
Vol 55 (14) ◽  
pp. 2685-2691 ◽  
Author(s):  
J. Bryan Jones ◽  
H. Bruce Goodbrand
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Primary Alcohol ◽  
Horse Liver Alcohol Dehydrogenase ◽  
Preparative Scale ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver

Horse liver alcohol dehydrogenase has been shown to be effective in catalyzing regiospecific oxidations of only the primary alcohol functions of several cyclohexane substrates possessing both primary and secondary alcohol substituents. The reactions, which were all performed on a preparative scale, were also enantioselective in some cases.

scholarly journals Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA, Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

2016 ◽  
Vol 199 (3) ◽  
Author(s):  
Tianyong Zheng ◽  
Daniel G. Olson ◽  
Sean J. Murphy ◽  
Xiongjun Shao ◽  
Liang Tian ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Single Gene ◽  
Maximum Yield ◽  
Primary Alcohol ◽  
Content Type ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Dehydrogenase Gene ◽  
High Ethanol

ABSTRACT Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at about 90% of the theoretical maximum yield (2 ethanol molecules per glucose equivalent) and a titer of 70 g/liter. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here, we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ∼50%, whereas multiple gene deletions of all annotated alcohol dehydrogenase genes except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked alcohol dehydrogenase (ADH) activity (acetaldehyde-reducing direction) by 93%. IMPORTANCE In this study, we set out to identify the alcohol dehydrogenases necessary for high ethanol production in T. saccharolyticum. Based on previous work, we had assumed that adhE was the primary alcohol dehydrogenase gene. Here, we show that both adhA and adhE are needed for high ethanol yield in the engineered strain LL1049. This is the first report showing adhA is important for ethanol production in a native adhA host, which has important implications for achieving higher ethanol yields in other microorganisms.

scholarly journals Cloning, sequencing and expression inEscherichia coliof the primary alcohol dehydrogenase gene fromThermoanaerobacter ethanolicusJW200

2000 ◽  
Vol 190 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Peter J Holt ◽  
Richard E Williams ◽  
Keith N Jordan ◽  
Christopher R Lowe ◽  
Neil C Bruce
Keyword(s):  
Alcohol Dehydrogenase ◽  
Primary Alcohol ◽  
Dehydrogenase Gene ◽  
Sequencing And Expression

scholarly journals cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions

1999 ◽  
Vol 65 (12) ◽  
pp. 5242-5246 ◽  
Author(s):  
Henning Raschke ◽  
Thomas Fleischmann ◽  
Jan Roelof Van Der Meer ◽  
Hans-Peter E. Kohler
Keyword(s):  
Escherichia Coli ◽  
Alcohol Dehydrogenase ◽  
Pseudomonas Sp ◽  
Dihydrodiol Dehydrogenase ◽  
Substituted Toluenes

ABSTRACT cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) fromPseudomonas sp. strain P51, cloned into Escherichia coli DH5α(pTCB149) was able to oxidizecis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and fourpara-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture ofcis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (−)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers ofcis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (−)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures ofpara-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.

scholarly journals ntn Genes Determining the Early Steps in the Divergent Catabolism of 4-Nitrotoluene and Toluene in Pseudomonas sp. Strain TW3

1998 ◽  
Vol 180 (8) ◽  
pp. 2043-2049 ◽  
Author(s):  
Keith D. James ◽  
Peter A. Williams
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Open Reading Frame ◽  
Pseudomonas Sp ◽  
Tol Plasmid ◽  
Reading Frame ◽  
Toluene Monooxygenase ◽  
Dehydrogenase Gene ◽  
Amino Acid Levels ◽  
Benzaldehyde Dehydrogenase

ABSTRACT Pseudomonas sp. strain TW3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the TOL plasmids. We report the sequence and organization of five genes, ntnWCMAB*, which are very similar to and in the same order as the xyl operon of TOL plasmid pWW0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respectively. These genes encode an alcohol dehydrogenase homolog (ntnW), an NAD+-linked benzaldehyde dehydrogenase (ntnC), a two-gene toluene monooxygenase (ntnMA), and part of a benzyl alcohol dehydrogenase (ntnB*), which have 84 to 99% identity at the nucleotide and amino acid levels with the corresponding xylWCMABgenes. The xylB homolog on the TW3 genome (ntnB*) appears to be a pseudogene and is interrupted by a piece of DNA which destroys its functional open reading frame, implicating an additional and as-yet-unidentified benzyl alcohol dehydrogenase gene in this pathway. This conforms with the observation that the benzyl alcohol dehydrogenase expressed during growth on 4-nitrotoluene and toluene differs significantly from the XylB protein, requiring assay via dye-linked electron transfer rather than through a nicotinamide cofactor. The further catabolism of 4-nitrobenzoate and benzoate diverges in that the former enters the hydroxylaminobenzoate pathway as previously reported, while the latter is further metabolized via the β-ketoadipate pathway.

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