The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 Å resolution by X-ray crystallography

Benach, J.; Atrian, Sı́lvia; Gonzàlez‐Duarte, Roser; Ladenstein, Rudolf

doi:10.1006/jmbi.1999.2765

Cited by 96 publications

(93 citation statements)

References 89 publications

(143 reference statements)

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“…2A), consisting of a central ␤-sheet surrounded by ␣-helices and a typical nicotinamide coenzyme binding ␤␣␤␣␤ subdomain with a characteristic Gly-Xaa-Gly-Xaa-Xaa-Gly motif (position 13-18). Asp-37 confers specificity toward NAD binding, whereas the active site is characterized by a Ser-Tyr-Lys catalytic triad (12)(13)(14). These and other conserved SDR features are preserved in JGW, as shown in Fig.…”

Section: Resultsmentioning

confidence: 90%

“…2B) (12, 13). Two replacements, His-191 3 Gln (His191Gln) and Glu-205 3 Lys (Glu205Lys), lie between Thr-186 and Pro-210, a region that forms the flap guarding the entrance to the active site and that is known to be responsible for the different specificities among related enzymes (12,13). Two additional replacements, Ser215Pro and Leu120Met, contact residues within the Thr-186-Pro-210 region and also may influence specificity.…”

Section: Resultsmentioning

confidence: 99%

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Evolving protein functional diversity in new genes of Drosophila

Zhang

Dean

Brunet

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

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The mechanism by which protein functional diversity expands is an important evolutionary issue. Studies of recently evolved chimeric genes permit direct investigation of the origin of new protein functions before they become obscured by subsequent evolution. Found in several African Drosophila species, jingwei (jgw), a recently evolved gene with a domain derived from the still extant short-chain alcohol dehydrogenase (ADH) through retroposition, provides an opportunity to examine this previously undescribed process directly. We expressed JGW proteins in a microbial expression system and, after purification, investigated their enzymatic properties. We found that, unexpectedly, positive Darwinian selection for amino acid replacements outside the active site of JGW produced a novel dehydrogenase with altered substrate specificity compared with the ancestral ADH. Instead of detoxifying and assimilating ethanol like its Adh parental gene, we observe that JGW efficiently utilizes long-chain primary alcohols found in hormone and pheromone metabolism. These data suggest that protein functional diversity can expand rapidly under the joint forces of exon shuffling, gene duplication, and natural selection.

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Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Evolving protein functional diversity in new genes of Drosophila

Zhang

Dean

Brunet

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

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“…DHRS6 shows an unusual sequence motif (TAAAQGIG instead of TGXXXGIG found in the majority of SDRs), which determines the turn between ␤A and ␣B, necessary to accommodate the pyrophosphate moiety of the cofactor (6,13,14). The degree of variability in this basic and critical SDR motif is also observed in Drosophila ADH, which has the sequence VAALGIG (15).…”

Section: Cofactor Binding and Active Site Architecture Of Human Dhrs6-mentioning

confidence: 99%

Characterization of Human DHRS6, an Orphan Short Chain Dehydrogenase/Reductase Enzyme

Guo

Lukacik

Papagrigoriou

et al. 2006

Journal of Biological Chemistry

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Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD ؉ -dependent conversion of (R)-hydroxybutyrate to acetoacetate with K m values of about 10 mM, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 Å in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg 144 , Arg 188 , and Arg 205 ) was found to bind a sulfate molecule at the active site. Docking analysis of R-␤-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.Hepatic ketone body formation and utilization of these compounds by peripheral tissues with high energy demands is essential in humans and other mammals for survival during times of starvation and extended fasting. The compounds categorized as ketone bodies comprise acetoacetate, R-␤-hydroxybutyrate, and acetone, the last being a nonmetabolizable decarboxylation product of acetoacetate. The liver produces and excretes ketone bodies during times when the amount of acetyl-CoA exceeds the oxidative capacity of hepatic mitochondria. Ketone body formation is thus necessary to maintain ␤-oxidation by supplying free CoA. This metabolic situation occurs when high amounts of fatty acids derived from peripheral tissues during starvation or fasting are oxidized by ␤-oxidation pathways. In exacerbated disease states, such as ketoacidotic coma, extremely high amounts of fatty acids are oxidized as a consequence of insulin resistance in diabetes mellitus. In this situation, a potentially life-threatening metabolic acidosis occurs through the high amounts of protons provided by acetoacetate and R-␤-hydroxybutyrate, exceeding the serum bicarbonate buffer capacity. Serum levels of 5-10 mM ketone bodies are reached under these circumstances as compared with levels of 1 mM in normal states. The liver synthesizes acetoacetate through the enzymes thiolase, hydroxymethylglutaryl-CoA synthase, and hydroxymethylglutaryl-CoA lyase from three molecules of acetyl-CoA, thus producing 1 mol of acetoacetate, 1 mol of acetyl-CoA, and 2 mol of free CoA. Acetoacetate is further reduced to R-␤-hydroxybutyrate through mito...

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“…S1). They feature a flexible substrate-binding loop spanning roughly residues 190-210, the most variable portion of SDRs, which has been implicated as crucial in determining the stereoselectivity of KREDs (14). In its closed conformation, this motif flanks one side of the active site and closes around the bound cofactor and substrate (11).…”

mentioning

confidence: 99%

Origins of stereoselectivity in evolved ketoreductases

Noey

Tibrewal

Jiménez‐Osés

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

108

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Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo-and NADP + -bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation. directed evolution | crystallographic structures | molecular dynamics | theozyme | enantioselectivity B iocatalysis is a common method of stereoselective ketone reduction (1). This approach often replaces multistep syntheses and uses renewable, biodegradable, and nontoxic reagents and mild conditions (2). Ketoreductases (KREDs), the most commonly used enzymes in industrial pharmaceutical synthesis (3), reduce a wide range of ketones to alcohols with high chemoselectivity and stereoselectivity. These enzymes have been engineered to synthesize alcohols as intermediates for the production of atorvastatin (Lipitor), montelukast (Singulair), and atazanavir (Reyetaz) (4).Small and almost symmetrical ketones, such as prochiral cyclopentanones, are attractive substrates that are difficult to reduce asymmetrically by chemical methods (5, 6). In particular, the enantiopure chiral alcohols derived from 3-oxacyclopentanone (1) and 3-thiacyclopentanone (2) are used in the synthesis of the pharmaceutical agents fosamprenavir and sulopenem, respectively (Fig. 1). Through a directed evolution (DE) program, Codexis, Inc. engineered a KRED obtained from Lactobacillus kefir for the reduction of 3-thiacyclopentanone (2) for the large-scale production of the antibiotic sulopenem. L. kefir KRED (WT) belongs to the short-chain dehydrogenase/reductase (SDR) family (7,8). Via DE, a variant containing 10 mutati...

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The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 Å resolution by X-ray crystallography

Cited by 96 publications

References 89 publications

Evolving protein functional diversity in new genes of Drosophila

Evolving protein functional diversity in new genes of Drosophila

Characterization of Human DHRS6, an Orphan Short Chain Dehydrogenase/Reductase Enzyme

Origins of stereoselectivity in evolved ketoreductases

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