Proc. Natl. Acad. Sci. USAVol. 93, pp. 5595-5599, May 1996BiochemistryPea formaldehyde-active class III alcohol dehydrogenase:Common derivation of the plant and animal forms but notof the corresponding ethanol-active forms (classes I and P)(pea enzyme structure/alcohol dehydrogenase origin/separate duplications/formaldehyde dehydrogenase/parallel evolution)JAWED SHAFQAT*, MUSTAFA EL AHMAD*, OLLE DANIELSSON*, M. CARMEN MART1NEZt, BENGT PERSSON*,XAVIER PARESt, AND HANs J6RNVALL**Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden; and tDepartment of Biochemistry and MolecularBiology, Universitat Autonoma de Barcelona, 08193 Bellaterra (Barcelona), SpainCommunicated by Sune Bergstrom, Karolinska Institutet, Stockholm, Sweden, January 4, 1996 (received for review November 28, 1995)ABSTRACT A plant class III alcohol dehydrogenase (orglutathione-dependent formaldehyde dehydrogenase) hasbeen characterized. The enzyme is a typical class III memberwith enzymatic parameters and substrate specificity closelyrelated to those of already established animal forms. Km valueswith the pea enzyme are 6.5 ,LM for NADI), 2 ,uM forS-hydroxymethylglutathione, and 840 ,uM for octanol versus9, 4, and 1200 ftM, respectively, with the human enzyme.Structurally, the pea/human class III enzymes are closelyrelated, exhibiting a residue identity of 69%So and with only 3 of23 residues differing among those often considered in sub-strate and coenzyme binding. In contrast, the correspondingethanol-active enzymes, the long-known human liver and peaalcohol dehydrogenases, differ more (47% residue identities)and are also in functionally important active site segments,with 12 of the 23 positions exchanged, including no less than7 at the usually much conserved coenzyme-binding segment.These differences affect functionally important residues thatare often class-distinguishing, such as those at positions 48,51, and 115, where the plant ethanol-active forms resembleclass III (Thr, Tyr, and Arg, respectively) rather than theanimal ethanol-active class I forms (typically Ser, His, andAsp, respectively). Calculations of phylogenetic trees supportthe conclusions from functional residues in subgrouping plantethanol-active dehydrogenases and the animal ethanol-activeenzymes (class I) as separate descendants from the class IIIline. It appears that the classical plant alcohol dehydroge-nases (now called class P) have a duplicatory origin separatefrom that of the animal class I enzymes and therefore aparalogous relationship with functional convergence of theiralcohol substrate specificity. Combined, the results establishthe conserved nature of class III also in plants, and contributeto the molecular and functional understanding of alcoholdehydrogenases by defining two branches of plant enzymesinto the system.Different sets of dimeric zinc-containing alcohol dehydroge-nases of the medium-chain dehydrogenase/reductase (MDR)(1) type have been characterized in animals and plants. One setencompasses animal alcohol dehydrogenases, including theclassical, ethanol-active liver enzyme of class I with about 20characterized enzymes (2-5), the apparently parent (6, 7) classIII form [or glutathione-dependent formaldehyde dehydroge-nase, present also in prokaryotes (8, 9)], and a total ofminimally six classes (10) and some mixed-class lines (11, 12)in vertebrates. The other is the set of plant alcohol dehydro-genases, of which about 20 enzymes have been structurallycharacterized (3, 4, 13). Further, MDR alcohol dehydroge-nases, but of a tetrameric type (including the yeast enzyme), aswell as other alcohol dehydrogenases (including the short-chain dehydrogenase/reductase, SDR, forms), also exist (seeref. 14). The plant and animal alcohol dehydrogenases, al-though definitely related (4, 15), raise questions about the classIII forms in plants and about the interrelationships of theethanol-active forms in plants and animals.The two sets of animal and plant ethanol-active enzymeswere initially compared (15) before knowledge of the enzymesystem at large and of the repeated gene duplications in theanimal line. The latter have been traced to early vertebrateevolution, with class III as the ancestral type, as supported byestimates of the divergence rate (16), the presence of mixed-class vertebrate forms presumably reflecting the enzymogen-esis (11, 12), and the presence of class III forms in invertebrates(17, 18) and prokaryotes (8, 9). In short, the animal enzymesystem appears to originate from class III, which is hardlyethanol-active, and to have evolved into the other classes,including the ethanol-active class I liver enzyme, during ver-tebrate radiation. However, the structurally characterizedplant enzymes exhibit reasonable ethanol activity, like the classI animal enzymes, and structural similarity to the class Iproteins (4, 15). Furthermore, although a plant class IIIenzyme exists and has been partially purified as a formalde-hyde dehydrogenase activity (19), no class III plant enzyme hasbeen structurally characterized. The apparently ancestral classIII line of the animal set has not been similarly defined in theplant set, in spite of the early class III origin. At the same time,the vertebrate ethanol-active classes of supposedly later originappear to have equivalents in the plant line, which, if theyreflect direct descendance from a common origin, wouldsuggest an earlier origin than that postulated from the patternof the animal classes alone. Clearly, this complicates theevolutionary scheme: either class III would be expected to bea distant ancestor also in the plant line and the vertebrate-specific enzyme classes not to be codescendants in one cladewith the ethanol-active plant line, or the origin of the animalforms has to be reconsidered to include the conclusions fromthe plant enzymes. The unresolved relationships questioninterpretations on the origins and functions of the eukaryoticalcohol dehydrogenases at large, also making this complexenzyme system unclear in humans.We have now characterized a plant class III enzyme frompea. Both its enzymatic and structural properties are clearlyrelated to those of the class III enzyme of other sources,establishing that this class is universally constant in all lifeforms. The structure further establishes the relationshipsamong the ethanol-active lines. They exhibit parallel evolu-Abbreviation: MDR, medium-chain dehydrogenase/reductase.Data deposition: The sequence reported in this paper has beendeposited in the Swissprot data base (accession no. P80572).5595The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.
