Amphibian Alcohol Dehydrogenase

Peralba, Josep Maria; Crosas, Bernat; Martínez, Sergi Robles; Julià, Pere; Farrés, Jaume; Parés, Xavier

doi:10.1007/978-1-4615-4735-8_42

Cited by 2 publications

(4 citation statements)

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“…3). Moreover, the k cat /K m values with NAD(H) obtained in the triple mutant are similar to those of typical NAD-dependent ADHs (37)(38)(39)(40)(41)(42). It is conceivable that some kind of threshold in catalytic power has been attained in many ADHs regarding coenzyme usage.…”

Section: Effect Of Substitutions On the Rate-limitingsupporting

confidence: 62%

Complete Reversal of Coenzyme Specificity by Concerted Mutation of Three Consecutive Residues in Alcohol Dehydrogenase

Rosell

Valencia

Ochoa

et al. 2003

Journal of Biological Chemistry

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Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP min؊1 ) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.Coenzyme specificity is an important property of NAD(P)-dependent oxidoreductases that is linked to their metabolic function. Thus the type of coenzyme, NAD ϩ or NADP ϩ , often distinguishes between enzymes involved in alternative pathways (e.g. oxidative versus reductive or degradative versus biosynthetic). Because NAD ϩ and NADP ϩ only differ structurally in the phosphate group esterified at the 2Ј position of adenosine ribose, dehydrogenases must possess a limited number of residues to discriminate between the two coenzyme types. Moreover, among dehydrogenases from a given enzyme family, the same protein fold is often used to bind either coenzyme type and even some enzymes show dual activity, meaning that they can use both coenzymes with similar efficiency (1).A rather unique NADP-dependent alcohol dehydrogenase (ADH8) 1 was discovered in the gastric tissues of amphibians (2). ADH8 belongs to the medium chain dehydrogenase/reductase (MDR) superfamily and is phylogenetically related to the NAD-dependent vertebrate ADH family. This enzyme is active with ethanol and functionally may participate in the reduction of retinal to retinol (k cat /K m all-trans-retinal ϭ 33,750 mM Ϫ1 min Ϫ1 ). Recently, the three-dimensional structure of the ADH8-NADP ϩ binary complex was determined at 1. define a binding pocket for the terminal phosphate group of NADP(H).Henceforth residue numbering will correspond to that of horse ADH1 with the Swiss Prot entry P00327. Interestingly, NADPdependent ADHs from distantly related microorganisms (5-7), also have a glycine and two more hydrophilic residues at the positions corresponding to 223, 224, and 225, respectively. In ADHs, residue 223 is located at the C-terminal end of the second ␤-strand of the Rossmann fold (8) and classically is considered as determinant for coenzyme specificity. The substitution D223G, as found in ADH8, would avoid the possible steric and electrostatic hindrances because of the extra phosphate group of NADP(H). In fact, different attempts to switch the coenzyme specificity in medium chain ADHs have been focused on mutations involving residue 223 (9 -12). However, full reversal of coenzyme specificity, in terms of having a mutant enzyme as catalytically efficient as the wild type, has been rarely achieved. This implies that conversion of coenzyme specificity may require multiple substitutions in the coenzymebinding domain. Other residues found in ADH8, such as Thr 224 and His 225 , which are making hydrogen bonds with the oxygen atoms from the terminal phosphate group (3), could also be important in defining co...

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Section: Effect Of Substitutions On the Rate-limitingsupporting

confidence: 62%

Complete Reversal of Coenzyme Specificity by Concerted Mutation of Three Consecutive Residues in Alcohol Dehydrogenase

Rosell

Valencia

Ochoa

et al. 2003

Journal of Biological Chemistry

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“…Xenopus ADH1A and ADH1B show Arg47, His51, Phe93 and Phe140, typical class I residues that are associated with ethanol dehydrogenase activity. The substrate-binding pocket of R. perezi ADH1B is extremely hydrophobic and space-restricted, resulting in low K m values for aliphatic alcohols, it has wide substrate specificity and is moderately active with retinoids [18]. In ADH1A, smaller substrate-binding residues Val141 and Val294 anticipate higher K m values for this isozyme, and substitution by His363 (Arg in many class I enzymes) suggests an increased rate of NAD + dissociation and higher k cat values.…”

Section: Discussionmentioning

confidence: 99%

“…ADH3 is a glutathione-dependent formaldehyde dehydrogenase, as seen spectrophotometrically for the purified enzyme of R. perezi [18] and by activity staining of electrophoresed tissue homogenates from R. perezi and X. laevis . Expression of amphibian ADH3 is detected in every stage and tissue studied, although it is more abundant in some organs such as the ovary, suggesting that oocytes may store large amounts of maternal ADH3 for its later use during the embryonic development.…”

Section: Discussionmentioning

confidence: 99%

“…Isozyme patterns of X. laevis liver ethanol dehydrogenase suggested the existence of two polymorphic genes encoding ADH subunits that did not form heterodimers and were located in different linkage groups [16,17]. The enzymes ADH1, ADH3 and ADH8 from the frog Rana perezi were purified and characterized by our group, and the ADH1 and ADH8 proteins were also sequenced [15,18]. The cloning of the cDNA of R. perezi ADH8 [15] allowed to perform mutagenesis studies on coenzyme specificity [19] and to obtain the crystal structure of the enzyme [20,21].…”

Section: Introductionmentioning

confidence: 99%

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The Xenopusalcohol dehydrogenase gene family: characterization and comparative analysis incorporating amphibian and reptilian genomes

et al. 2014

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BackgroundThe alcohol dehydrogenase (ADH) gene family uniquely illustrates the concept of enzymogenesis. In vertebrates, tandem duplications gave rise to a multiplicity of forms that have been classified in eight enzyme classes, according to primary structure and function. Some of these classes appear to be exclusive of particular organisms, such as the frog ADH8, a unique NADP+-dependent ADH enzyme. This work describes the ADH system of Xenopus, as a model organism, and explores the first amphibian and reptilian genomes released in order to contribute towards a better knowledge of the vertebrate ADH gene family.ResultsXenopus cDNA and genomic sequences along with expressed sequence tags (ESTs) were used in phylogenetic analyses and structure-function correlations of amphibian ADHs. Novel ADH sequences identified in the genomes of Anolis carolinensis (anole lizard) and Pelodiscus sinensis (turtle) were also included in these studies. Tissue and stage-specific libraries provided expression data, which has been supported by mRNA detection in Xenopus laevis tissues and regulatory elements in promoter regions. Exon-intron boundaries, position and orientation of ADH genes were deduced from the amphibian and reptilian genome assemblies, thus revealing syntenic regions and gene rearrangements with respect to the human genome. Our results reveal the high complexity of the ADH system in amphibians, with eleven genes, coding for seven enzyme classes in Xenopus tropicalis. Frogs possess the amphibian-specific ADH8 and the novel ADH1-derived forms ADH9 and ADH10. In addition, they exhibit ADH1, ADH2, ADH3 and ADH7, also present in reptiles and birds. Class-specific signatures have been assigned to ADH7, and ancestral ADH2 is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian ADH2 but with class-I kinetic properties. Remarkably, many ADH1 and ADH7 forms are observed in the lizard, probably due to lineage-specific duplications. ADH4 is not present in amphibians and reptiles.ConclusionsThe study of the ancient forms of ADH2 and ADH7 sheds new light on the evolution of the vertebrate ADH system, whereas the special features showed by the novel forms point to the acquisition of new functions following the ADH gene family expansion which occurred in amphibians.

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Amphibian Alcohol Dehydrogenase

Cited by 2 publications

References 26 publications

Complete Reversal of Coenzyme Specificity by Concerted Mutation of Three Consecutive Residues in Alcohol Dehydrogenase

Complete Reversal of Coenzyme Specificity by Concerted Mutation of Three Consecutive Residues in Alcohol Dehydrogenase

The Xenopusalcohol dehydrogenase gene family: characterization and comparative analysis incorporating amphibian and reptilian genomes

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