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

Abstract

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.

New findings on the genetic influences on alcohol use and dependence

“…The newly identified ADH1C Ã 3 allele among Native Americans has not been enzymically characterized [82]. Recently, functional polymorphisms were identified in the coding region of the ADH2 gene and in the promoter region of the ADH4 gene [83,84].…”

Oxidation of celecoxib by polymorphic cytochrome P450 2C9 and alcohol dehydrogenase

“…Human ADH1C2 (previously class I, γ 2 γ 2 ), ADH2 (class II, ππ ) [14] and ADH3 (class III, χχ ) [15] were expressed in E. coli and purified in two steps with DEAE cellulose (Whatman) and either AMP sepharose or blue sepharose (Amersham Biosciences), as described earlier [16]. Proteins were concentrated on 30K cut-off filters in either centrifugal cells (centriplus YM-30, Millipore) or in a stirred ultrafiltration cell (Amicon/Millipore).…”

“…the formation of aldehyde metabolite, at 340 nm using a NADH absorption coefficient (ε) of 6220 M −1 cm −1 . Steady-state enzyme kinetics were performed at 25 °C in 0.1 M glycine/NaOH, pH 10.0 or 0.1 M sodium phosphate, pH 7.5 with 2.4 mM NAD + [14]. The protein concentration was 0.87 mg ml −1 .…”

Oxidation of alcohols and reduction of aldehydes derived from methyl- and dimethylpyrenes by cDNA-expressed human alcohol dehydrogenases

“…Therefore, it was necessary for kinetic analyses to separate the cDNA-expressed human ADHs from the endogenous activity. For this separation we exploited the observation that various mammalian ADHs show unusually weak interactions with anion exchange materials (Strömberg et al, 2002). The separations were conducted by fast-performance liquid chromatography essentially as previously described for ADH2 (Kollock et al, 2008).…”

“…They differ in their substrate specificity, interactions with inhibitors and tissue distribution. Genetic polymorphisms leading to amino acid exchanges have been reported for ADH1B, ADH1C, ADH2 and ADH4 (Duester et al, 1999;Buervenich et al, 2000;Strömberg et al, 2002). For the specification of susceptibility factors, we are striving to identify the human ADH forms involved in the detoxification of benzylic alcohols of PAHs.…”