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

Abstract

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...

Biochemical Analysis of Halophilic Dehydrogenases Altered by Site-Directed Mutagenesis

“…In contrast with our results, in alcohol dehydrogenase from gastric tissues of Rana perezi, the complete reversal of coenzyme specificity from NADP(H) to NAD(H) was reached with the concerted mutation of three residues G223D/T224I/H225N (Rosell et al, 2003) . The single mutation G223D had no effect on catalysis with NAD + and the double mutant G223D/T224I was a better catalyst than the single mutant with NAD + , but worse than the triple mutant.…”

“…The great increase in the K cat observed with the GlcDH double mutant was not observed in alcohol dehydrogenase. It appears that one or two substitutions in alcohol dehydrogenase were not sufficient to transform coenzyme specificity, whereas multiple substitutions could be effective (Rosell et al, 2003). The same, but reverse, effect was observed with NAD + -dependent xylitol dehydrogenase, in that the reverse of coenzyme specificity from NAD + to NADP + was achieved with the triple mutant D207A/I208R/F209S (Watanabe et al, 2005).…”

Merging protein, gene and genomic data: the evolution of the MDR-ADH family

“…Although initially ascribed to class IV on the basis of its substrate specificity and gastric localization, as mammalian class IV forms, the three-dimensional structure (Rosell et al, 2003b;Valencia et al, 2003), in vitro behaviour -more as a retinal reductase than alcohol dehydrogenase -and its unique preference among vertebrate ADHs for NADP þ rather than NAD þ , all suggested a separate class, named class VIII. It has been proposed that the triad Gly223-Thr224-His225, together with Leu200 and Lys228, may account for the cofactor preference, also verified by sitedirected mutagenesis at the triad segment (Rosell et al, 2003a). Interestingly, in Xenopus, we have derived sequences that are highly similar to R. perezy ADH8: XlADH8 (73.1% similarity), XtADH8A (71.7%) and XtAHD8B (66.7%) (Table 1 and Figure 6).…”

Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis

“…Several attempts to alter the coenzyme specificity have been made for several dehydrogenases, and complete reversal was accomplished in xylitol dehydrogenase from Pichia stipitis and Saccharomyces cerevisiae (Watanabe et al, 2005;Krahulec et al, 2012), alcohol dehydrogenase from Rana perezi (Rosell et al, 2003) and lactate dehydrogenases from T. flavus (Tomita et al, 2006a). However, the information on alteration of coenzyme specificity in bacterial MDHs is scarce, except for TfMDH.…”