Enzymic and Structural Studies on Drosophila Alcohol Dehydrogenase and Other Short-Chain Dehydrogenases/Reductases

Smilda, Tim; Kamminga, A.; Reinders, Peter; Baron, Wia; Vlieg, Johan E. T. van Hylckama; Beintema, Jaap J.

doi:10.1007/s002390010175

Cited by 9 publications

(11 citation statements)

References 27 publications

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“…Haloalcohol dehalogenases, also known as halohydrin dehalogenases or halohydrin hydrogen‐halide lyases, cannot be classified in these existing dehalogenase families. Instead, they show low sequence similarity to members of the short‐chain dehydrogenase/reductase (SDR) family (Smilda et al ., 2001; van Hylckama Vlieg et al ., 2001). This family contains redox enzymes that depend on NAD(P)H, which is bound in a characteristic dinucleotide binding fold (Rossmann fold) (Rossmann et al ., 1974).…”

Section: Introductionmentioning

confidence: 99%

Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site

et al. 2003

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Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic. These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen. Haloalcohol dehalogenases are related to the widespread family of NAD(P)Hdependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones. Our results reveal the ®rst structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site. Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors. Keywords: haloalcohol dehalogenase/SDR family/shortchain dehydrogenase/X-ray structure IntroductionDehalogenases are enzymes that are able to cleave carbonhalogen bonds . Structural characterization of haloalkane dehalogenases and haloacid dehalogenases demonstrated that these hydrolytic enzymes are evolutionarily related to widespread esterase and phosphatase families (Ollis et al., 1992;Hisano et al., 1996;Ridder and Dijkstra, 1999). Haloalcohol dehalogenases, also known as halohydrin dehalogenases or halohydrin hydrogen-halide lyases, cannot be classi®ed in these existing dehalogenase families. Instead, they show low sequence similarity to members of the short-chain dehydrogenase/reductase (SDR) family . This family contains redox enzymes that depend on NAD(P)H, which is bound in a characteristic dinucleotide binding fold (Rossmann fold) (Rossmann et al., 1974). They have a conserved catalytic triad of Ser, Tyr and Lys/Arg residues (Jo Èrnvall et al., 1995;Oppermann et al., 2003), which is also present in the haloalcohol dehalogenases . Many structures of shortchain dehydrogenases/reductases in complex with dinucleotides and substrates have revealed the structural details of the reactions catalyzed by them (Jo Èrnvall et al., 1995;Filling et al., 2002;Oppermann et al., 2003). In addition, the structure of a dinucleotide-binding transcription factor that lacked the catalytic tyrosine and thus oxidoreductase activity showed that the SDR fold also functions in nonenzymatic activities (Stammers et al., 2001).Haloalcohol dehalogenases catalyze the intramolecular displacement of a halogen by the vicinal hydroxyl group in 1,3-dichloro-2-propanol, yielding its ...

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Section: Introductionmentioning

confidence: 99%

Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site

et al. 2003

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“…A number of structural studies have been performed on SDR enzymes from different sources, including mannitol-2-dehydrogenase from Agaricus bisporus (MtDH) (Hörer et al 2001), R-specific alcohol dehydrogenase from Lactobacillus brevis (RADH) (Schlieben et al 2005), b-ketoacyl reductase from E. coli (FabG) (Price et al 2001), and Drosophila alcohol dehydrogenase (ADH), the latter of which is the most widely characterized member of the SDR family in terms of substrate specificities (Benach et al 1999;Smilda et al 2001). Most SDR enzymes have a core structure of 250-350 residues in length and are often found in either dimeric or tetrameric forms (Jornvall et al 1995).…”

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confidence: 99%

Crystal structure of a carbonyl reductase from Candida parapsilosis with anti‐Prelog stereospecificity

et al. 2008

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A novel short-chain (S)-1-phenyl-1,2-ethanediol dehydrogenase (SCR) from Candida parapsilosis exhibits coenzyme specificity for NADPH over NADH. It catalyzes an anti-Prelog type reaction to reduce 2-hydroxyacetophenone into (S)-1-phenyl-1,2-ethanediol. The coding gene was overexpressed in Escherichia coli and the purified protein was crystallized. The crystal structure of the apo-form was solved to 2.7 Å resolution. This protein forms a homo-tetramer with a broken 2-2-2 symmetry. The overall fold of each SCR subunit is similar to that of the known structures of other homologous alcohol dehydrogenases, although the latter usually form tetramers with perfect 2-2-2 symmetries. Additionally, in the apo-SCR structure, the entrance of the NADPH pocket is blocked by a surface loop. In order to understand the structure-function relationship of SCR, we carried out a number of mutagenesisenzymatic analyses based on the new structural information. First, mutations of the putative catalytic Ser-Tyr-Lys triad confirmed their functional role. Second, truncation of an N-terminal 31-residue peptide indicated its role in oligomerization, but not in catalytic activity. Similarly, a V270D point mutation rendered the SCR as a dimer, rather than a tetramer, without affecting the enzymatic activity. Moreover, the S67D/H68D double-point mutation inside the coenzyme-binding pocket resulted in a nearly 10-fold increase and a 20-fold decrease in the k cat /K M value when NADH and NADPH were used as cofactors, respectively, with k cat remaining essentially the same. This latter result provides a new example of a protein engineering approach to modify the coenzyme specificity in SCR and short-chain dehydrogenases/reductases in general.

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“…Kinetic and structural studies on ADH in D. lebanonensis and D. simulans have provided an explanation for this unique characteristic by showing that the active site is bifurcated and ideally suited for secondary alcohols (Benach et al, 2001;Smilda et al, 2001). However, species such as D. mojavensis from Baja that are subjected to elevated concentrations of 2-propanol in their host cactus seem particularly susceptible to the inhibitory effects of acetone produced by oxidation of 2-propanol by ADH.…”

Section: Discussionmentioning

confidence: 94%

“…Drosophila ADH, a member of the short-chain dehydrogenase/ reductase (SDR) family of enzymes, differs from mammalian ADH in that it does not require zinc as a cofactor, does not breakdown methanol, and shows a preference for secondary alcohols, especially 2-propanol (Benach et al, 2001;Smilda et al, 2001). 2-propanol, however, has been termed a ''suicide'' substrate (e.g.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of alcohol dehydrogenase after 2-propanol exposure in different geographic races ofDrosophila mojavensis: Lack of evidence for selection at theAdh-2 Locus

2005

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High frequencies of the fast allele of alcohol dehydrogenase-2 (Adh-2F) are found in populations of Drosophila mojavensis that inhabit the Baja California peninsula (race BII) whereas the slow allele (Adh-2S) predominates at most other localities within the species' geographic range. Race BII flies utilize necrotic tissue of pitaya agria cactus (Stenocereus gummosus) which contains high levels of 2-propanol, whereas flies from most other localities utilize different cactus hosts in which 2-propanol levels are low. To test if 2-propanol acts as a selective force on Adh-2 genotype, or whether some other yet undetermined genetic factor is responsible, mature males of D. mojavensis lines derived from the Grand Canyon (race A) and Santa Catalina Island (race C), each with individuals homozygous for Adh-2F and Adh-2S, were exposed to 2-propanol for 24 h and ADH-2 specific activity was then determined on each genotype. Flies from five other localities homozygous for either the fast or slow allele also were examined. Results for all reported races of D. mojavensis were obtained. 2-propanol exposure inhibited ADH-2 specific activity in both genotypes from all localities, but inhibition was significantly less in two populations of race BII flies homozygous for Adh-2F. When F/F and S/S genotypes in flies from the same locality were compared, both genotypes showed high 2-propanol inhibition that was not statistically different, indicating that the F/F genotype alone does not provide a benefit against the inhibitory effects of 2-propanol. ADH-1 activity in female ovaries was inhibited less by 2-propanol than ADH-2. These results do not support the hypothesis that 2-propanol acts as a selective factor favoring the Adh-2F allele.

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Enzymic and Structural Studies on Drosophila Alcohol Dehydrogenase and Other Short-Chain Dehydrogenases/Reductases

Cited by 9 publications

References 27 publications

Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site

Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site

Crystal structure of a carbonyl reductase from Candida parapsilosis with anti‐Prelog stereospecificity

Inhibition of alcohol dehydrogenase after 2-propanol exposure in different geographic races ofDrosophila mojavensis: Lack of evidence for selection at theAdh-2 Locus

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