The Crystal Structure of R-specific Alcohol Dehydrogenase from Lactobacillus brevis Suggests the Structural Basis of its Metal Dependency

Niefind, Karsten; Müller, Jörg; Riebel, Bettina R.; Hummel, Werner; Schomburg, Dietmar

doi:10.1016/s0022-2836(03)00081-0

Cited by 126 publications

(105 citation statements)

References 34 publications

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“…Moreover, incubation of the dialyzed enzyme at 30°C for 6 h in the presence of a low concentration of denaturant (0.5 M guanidinium HCl) resulted in 20% inactivation in the absence and presence of EDTA or o-phenanthroline added at millimolar concentrations (data not shown). These results indicate that ADH Tt does not require metals for activity or for structural stabilization; although this is usual for the typically nonmetal SDR enzymes, the more classical RADH Lb -type ADHs show strong Mg 2ϩ dependence (23). The 65% inactivation observed with the ionic liquid was presumably due to competition of the BF 4 Ϫ ion with the coenzyme phosphate moiety for the anion binding site of the enzyme.…”

Section: Vol 74 2008mentioning

confidence: 89%

Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus

Pennacchio

Pucci

Secundo

et al. 2008

Appl Environ Microbiol

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The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/ reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh Tt ) was heterologously overexpressed in Escherichia coli, and the protein (ADH Tt ) was purified to homogeneity and characterized. ADH Tt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ϳ73°C and a 30-min half-inactivation temperature of ϳ90°C, as well as good tolerance to common organic solvents. ADH Tt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and ␣-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, ␣-tetralone, and ␣-methyl and ␣-ethyl benzoylformates to (S)-(؊)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-␣-(trifluoromethyl)benzyl alcohol (93% ee), (S)-␣-tetralol (>99% ee), methyl (R)-(؊)-mandelate (92% ee), and ethyl (R)-(؊)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.Alcohol dehydrogenases (ADHs) are of interest for the synthesis of the (S) or (R) enantiomers of alcohols from prochiral ketones (11). Since their early application in asymmetric synthesis using horse liver and yeast ADHs (13) and Thermoanaerobacter brockii ADH (ADH Tb ) (15), screening efforts have been directed at various species of microorganisms, which has resulted in new ADHs that have distinctive substrate specificity, good efficiency, and high enantioselectivity. Representative examples of enzymes from mesophilic microorganisms are the NADP-dependent (R)-specific ADH from Lactobacillus brevis (RADH Lb ), which is active on aryl ketones and whose crystal structure has recently been solved (29), the NAD-dependent (S)-specific 1-phenylethanol dehydrogenase from the denitrifying bacterium strain EbN1 (PED), which was characterized and crystallized (10), and the NAD-dependent ADH from Leifsonia sp. strain S749 (ADH Ls ), which was found to be active on (R)-sec alcohols, aryl ketones, aldehydes, and keto esters and whose gene has recently been cloned for protein expression in Escherichia coli (12). These enzymes are homotetrameric and belong to the short-chain dehydrogenase/ reductase (SDR) superfamily (14), which is characterized by ϳ250-residue subunits, a Gly motif in the coenzyme-binding regions, and a catalytic triad formed by the highly conserved residues Tyr, Lys, and Ser, to which an Asn residue has been added based on the proposal of Filling et al. (2), which was...

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Section: Vol 74 2008mentioning

confidence: 89%

Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus

Pennacchio

Pucci

Secundo

et al. 2008

Appl Environ Microbiol

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“…Its high resolution crystal structure showed that the magnesium ion was bound by two glutamate residues per monomer to stabilize the dimer (20). In the second case, R-specific alcohol dehydrogenase from Lactobacillus brevis (Protein Data Bank code 1nxq) was shown to be a homotetramer stabilized by two structured magnesium ions per tetramer (21). The carboxylate of the C-terminal glutamine residue coordinates water molecules that bind magnesium.…”

Section: Resultsmentioning

confidence: 99%

Characterization of 5-Chloro-5-Deoxy-d-Ribose 1-Dehydrogenase in Chloroethylmalonyl Coenzyme A Biosynthesis

Kale

McGlinchey

Moore

2010

Journal of Biological Chemistry

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SalM is a short-chain dehydrogenase/reductase enzyme from the marine actinomycete Salinispora tropica that is involved in the biosynthesis of chloroethylmalonyl-CoA, a novel halogenated polyketide synthase extender unit of the proteasome inhibitor salinosporamide A. SalM was heterologously overexpressed in Escherichia coli and characterized in vitro for its substrate specificity, kinetics, and reaction profile. A sensitive real-time 13

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“…ChKRED20, predicted ketone reductase in this work, KC342020; LSADH, alcohol dehydrogenase from Leifsonia sp. S749, BAD99642 [31]; LK-ADH, R-specific alcohol dehydrogenase from Lactobacillus kefir, AAP94029 [28]; LB-RADH, R-specific alcohol dehydrogenase from Lactobacillus brevis, AJ544275 [32]; TtADH, alcohol dehydrogenase from Thermus thermophilus, 2D1Y [36]; CPADH, (S)-1-phenyl-1,2-ethanediol dehydrogenase from Candida parapsilosis, DQ675534 [29]. Gray shading indicates residues highly conserved in the SDR family.…”

Section: Discussionmentioning

confidence: 99%

“…All other SDRs shared <40% identity with ChKRED20 ( Table 2). Some of them, such as LB-RADH from Lactobacillus brevis, follow the anti-Prelog's rule [32], whereas others such as SaADH2 from Sulfolobus acidocaldarius follow Prelog's rule [33] (Table 2).…”

Section: Sequence Analysis Of Chkred20mentioning

confidence: 99%

Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone

Liu

Tang

Pei

et al. 2014

Journal of Molecular Catalysis B: Enzymatic

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The Crystal Structure of R-specific Alcohol Dehydrogenase from Lactobacillus brevis Suggests the Structural Basis of its Metal Dependency

Cited by 126 publications

References 34 publications

Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus

Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus

Characterization of 5-Chloro-5-Deoxy-d-Ribose 1-Dehydrogenase in Chloroethylmalonyl Coenzyme A Biosynthesis

Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone

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