Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria

Radianingtyas, Helia; Wright, Phillip C.

doi:10.1016/s0168-6445(03)00068-8

Cited by 106 publications

(84 citation statements)

References 96 publications

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“…The cell extract activity measurements in Table 2 suggest that this other ADH is NADPH linked and may have higher ADH activity than AdhE. It has been reported that an NADPH-linked primary ADH, AdhA, is present in the Thermoanaerobacter species and may be part of the ethanol production pathway (43,44). Sequence analysis shows that T. saccharolyticum JW/SL-YS485 has a gene (Tsac_2087) encoding an ADH that is 86% identical (at the protein level) to T. mathranii and T. ethanolicus AdhA.…”

Section: Discussionmentioning

confidence: 98%

Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum

et al. 2015

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Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (ϳ50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. IMPORTANCEThis work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production by C. thermocellum and T. saccharolyticum. T hermophilic organisms, Clostridium thermocellum in particular, hold great promise for the production of ethanol from lignocellulosic feedstocks (1, 2). C. thermocellum is a thermophilic, Gram-positive obligate anaerobe that rapidly consumes cellulose. Engineered strains of Thermoanaerobacterium saccharolyticum (3), a thermophilic anaerobe that ferments xylan and other sugars derived from biomass, have been shown to produce ethanol at Ͼ50 g/liter, a near-theoretical yield (4). While comparable concentrations of ethanol are tolerated by selected strains of C. thermocellum (5-7), the maximum reported concentration of ethanol produced by this organism is 23.6 g/liter (8) and th...

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Section: Discussionmentioning

confidence: 98%

Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum

et al. 2015

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“…Almost all are NAD(P) dependent and are classified into three different groups, the zinc-dependent ADHs, the short-chain ADHs, and the ironactivated ADHs (20). The enzyme described here, AdhD, represents a novel NAD(P)H-dependent alcohol dehydrogenase from Pyrococcus furiosus that does not show similarity with these groups but rather with the aldo-keto reductase superfamily.…”

Section: Discussionmentioning

confidence: 99%

Production and Characterization of a Thermostable Alcohol Dehydrogenase That Belongs to the Aldo-Keto Reductase Superfamily

Machielsen

Uria

Kengen

et al. 2006

Appl Environ Microbiol

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The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100°C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.

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“…Alcohol dehydrogenase (AlcDH) and aldehyde dehydrogenase (AldDH) catalyze the conversion of aldehydes to alcohols and carboxylic acids, respectively. The neighbor-joining trees of both the AlcDH and the AldDH families appear to be large and complicated (51,54). Since the experimental evidence of AlcDH and AldDH from LAB is lacking, only several putative AlcDH subfamilies and a bifunctional AlcDH/AldDH subfamily can be distinguished (data not shown).…”

Section: Enzymes Involved In the Transamination Routementioning

confidence: 99%

Comparative Genomics of Enzymes in Flavor-Forming Pathways from Amino Acids in Lactic Acid Bacteria

Liu

Nauta

Francke

et al. 2008

Appl Environ Microbiol

191

162

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4Lactic acid bacteria (LAB) have been widely used as starter or nonstarter cultures in the dairy industry for over a thousand years. They play an essential role in flavor formation during the fermentation of dairy products. Several metabolic routes can lead to the formation of flavor compounds when LAB are growing in milk. One of the main precursors for flavor compounds in milk is casein, although they can also be derived from fatty acids and sugars. The proteolytic system of LAB degrades casein into its constituent amino acids, which can be converted to flavor compounds. Although amino acid catabolism by LAB has been well researched (5, 31, 65, 78), many flavor-forming routes are yet to be discovered.Over 20 LAB genomes have been fully sequenced (1,11,12,16,19,41,44,45,53, 68,71). The available genomic information provides us with new opportunities to study the flavorforming potential of LAB. However, one of the main problems that one encounters while reconstructing flavor-forming routes based on the genomic information stored in the public databases is the inconsistency in the functional annotation for many of the relevant genes. These genes are mostly members of larger protein families. Moreover, even when the functional annotation in databases is appropriate, it sometimes reflects only part of the protein's full functional potential, since broad substrate specificities are often not taken into consideration in the annotation.We have now improved the functional annotation of the key enzymes in the formation of flavor compounds from amino acids by applying comparative genomics approaches that have been developed within our group to specify the annotation of homologous proteins, by combining phylogeny, gene context, and experimental evidence (32). We focused especially on the enzymes involved in the metabolism of sulfur-containing amino acids since these are known to be precursors of many flavor compounds in dairy fermentations. Comparative analysis of the various sequenced LAB species and strains resulted in an overall view of differences in their flavor-forming capacities.

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Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria

Cited by 106 publications

References 96 publications

Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum

Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum

Production and Characterization of a Thermostable Alcohol Dehydrogenase That Belongs to the Aldo-Keto Reductase Superfamily

Comparative Genomics of Enzymes in Flavor-Forming Pathways from Amino Acids in Lactic Acid Bacteria

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