Structure of the class II enzyme of human liver alcohol dehydrogenase: combined complementary DNA and protein sequence determination of the .pi. subunit

Jo, Höög; H, von Bahr-Lindström; Lo, Hedén; Holmquist, Barton; Larsson, Kerstin; Hempel, John; Bl, Vallee; Jörnvall, Hans

doi:10.1021/bi00381a021

Cited by 74 publications

(6 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The coding region of human ADH2 [28] was subcloned into the pET3d expression vector (Novagen) and sitedirected mutagenesis was performed on double-stranded plasmid isolated with the flexiprep kit (Amersham Pharmacia Biotech). The transition resulting in the Ile/Val substitution at position 308 was introduced using the U.S.E.…”

Section: Plasmid Constructs and Site-directed Mutagenesismentioning

confidence: 99%

“…A model of human ADH2 (Swissprot code ADHP_HU-MAN) [28,33] was created by homology modelling from the mouse enzyme (PDB code 1e3i) [27,34]. The template and the target showed 72 % sequence identity, which makes homology modelling feasible.…”

Section: Structure Modellingmentioning

confidence: 99%

“…In an attempt to confirm or dismiss four previously proposed polymorphisms in the ADH2 gene [28], PCR-amplified fragments of exons 7 and 9 (approximately 150 bp each) were analysed by single-strand conformation polymorphisms (SSCP) gel electrophoresis. For the exon 7 samples, three different migration patterns were readily detected ( fig.…”

Section: Identification Of a New Adh2 Allelementioning

confidence: 99%

“…The ADH2 promoter was found to contain three polymorphic sites that affected gene expression in a reporter gene assay [7]. Four additional polymorphisms have been proposed in the coding sequence of the human ADH2 gene based on discrepancy between peptide and cDNA sequences [28]. Two of these possible polymorphisms were in exon 7, corresponding to substitutions at positions 308 and 317 in the polypeptide chain, whereas the other two substitutions were in the ninth terminal exon, position 373 and the termination codon.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2

Strömberg

Svensson

Hedberg

et al. 2002

Cellular and Molecular Life Sciences (CMLS)

View full text Add to dashboard Cite

The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes. A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis. Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation. Thermostability was assayed by activity measurements and circular dichroism spectroscopy. The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage. Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308. Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance. In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.

show abstract

Section: Plasmid Constructs and Site-directed Mutagenesismentioning

confidence: 99%

Section: Structure Modellingmentioning

confidence: 99%

Section: Identification Of a New Adh2 Allelementioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2

Strömberg

Svensson

Hedberg

et al. 2002

Cellular and Molecular Life Sciences (CMLS)

View full text Add to dashboard Cite

show abstract

“…Enzymatic analysis of catalytic properties, gene expression pattern, and comparison of DNA sequences of ADHs in various vertebrate species have caused them to be classified them into seven distinct classes (Duester et al 1999). One of the family members, the human alcohol dehydrogenase 4 (ADH4) gene (OMIM: 103740), encodes a class II pi-ADH that catalyzes oxidoreduction of a broad range of alcohols and the corresponding aldehydes (Höög et al 1987;Ditlow et al 1984;Sellin et al 1991). The gene spans an approximately 21-kb genomic region and consists of nine exons (von BahrLindström et al 1991).…”

Section: Introductionmentioning

confidence: 99%

Thirteen single-nucleotide polymorphisms (SNPs) in the alcohol dehydrogenase 4 (ADH4) gene locus

et al. 2002

View full text Add to dashboard Cite

The human alcohol dehydrogenase 4 (ADH4) gene encodes the class II ADH4 pi subunit, which contributes to the metabolization of a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Here we report the results of systematic screening for singlenucleotide polymorphisms (SNPs) in the ADH4 gene by means of direct sequencing combined with a polymerase chain reaction method. A total of 16 genetic variations including 13 SNPs were found; 4 in the 5Ј flanking region, 4 in the 5Ј untranslated region, and 8 within introns. No variation was found in coding, 3Ј untranslated, or 3Ј flanking regions. Eight of the 13 SNPs were not reported in the NCBI dbSNP database or any previous publications. Our SNP map presented here should provide tools to evaluate the role of ADH4 in complex genetic diseases and a variety of pharmacogenetic effects.

show abstract

Zn‐Dependent Medium‐Chain Dehydrogenases/Reductases

Meijers

Cedergren-Zeppezauer

2004

Handbook of Metalloproteins

View full text Add to dashboard Cite

Medium‐chain dehydrogenases/reductases (MDRs) have about 350 to 390 amino acids and belong to a large superfamily of proteins including the Zn‐dependent alcohol dehydrogenases (ADHs) and several other Zn‐dependent activities. They are dimers or tetramers and utilize either NADH or NADPH as electron carriers referred to as cofactors. The oxidation/reduction process is a 2‐electron/1‐proton (hydride ion, H − ) transfer between substrate and cofactor. The compounds oxidized or reduced by these enzymes vary considerably in structure ranging from small primary and secondary alcohols/aldehydes/ketones (aliphatic and aromatic), sugars (sorbitol/fructose, glucose) to large substances like fatty acids, steroids, and retinoids. ADHs are widely distributed in nature and some MDR members are found in all species. An essential Zn ion at the active site is common to all MDRs but some members have two zinc ions per subunit. The 3D Structures of Zn‐dependent MDRs are highly similar and they essentially have the same polypeptide fold. The low sequence identity found sometimes could not immediately reveal this close structural relationship. Now numerous structures have been examined indicating that the evolutionary strategy for MDRs has been to vary amino acids preferably at the reaction center and further away in the substrate binding area. Thus, the size and shape of the substrate channel is adapted for small or large substrates, hydrophilic or hydrophobic substances, modifications attained via few mutations, or significant deletions/additions of amino acids. A further opportunity to create multiplicity has been to vary the control mechanism for the protein conformation change and the oligomeric state. Some enzymes undergo significant structural changes upon cofactor binding, while others do not. A transition state intermediate of NADH in complex with horse liver alcohol dehydrogenase, resolved to atomic resolution (≈1 Å), has revealed structural modifications of the cofactor associated with the activation process for hydride transfer. This involves an OH − pyridine ring adduct, connected to the zinc center. The distortion of the nicotinamide at the enzyme site, affected by the presence of the negatively charged oxygen ligand, influences three parameters: (i) the puckering of the ring, (ii) a deviation from a standard carbon–carbon double bond length, and (iii) accumulation of negative charge at the C4 atom promoting hydride transfer. The active site Zn‐center of this large enzyme group shows great flexibility with respect to metal–ligand geometry and dynamics. Structural and spectroscopic evidence suggest that Zn‐dependent MDRs should be included in the general family of Zn‐enzymes, which require water and hydroxide ion as reactants during catalysis.

show abstract

Structure of the class II enzyme of human liver alcohol dehydrogenase: combined complementary DNA and protein sequence determination of the .pi. subunit

Cited by 74 publications

References 54 publications

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2

Thirteen single-nucleotide polymorphisms (SNPs) in the alcohol dehydrogenase 4 (ADH4) gene locus

Zn‐Dependent Medium‐Chain Dehydrogenases/Reductases

Contact Info

Product

Resources

About