Proc. Nati. Acad. Sci. USAVol. 91, pp. 4980-4984, May 1994Biochemistry

Fundamental molecular differences between alcoholdehydrogenase classes

(Drosophila octano dehydrogenase/class m alcohol dehydrogenase/mo ur patterns/zinc enyme famy)

OLLE DANIELSSON*, SILVIA ATRIANt, TERESA LUQUEt, LARS HJELMQVIST*, ROSER GONZALEZ-DUARTEt,AND HANS J6RNVALL*f*Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden; tCenter for Biotechnology, Karolinska Institutet,S-141 86 Huddinge, Sweden; and tDepartment of Genetics, University of Barcelona, E-08071 Barcelona, Spain

Communicated by Sune Bergstrom, January 18, 1994

ABSTRACT Two types of alcohol dehydrogenase in sepa-rate protein families are the "medium-chain" zinc enzymes(including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although themedium-chain family has been characterized in prokaryotesand many eukaryotes (fungi, plants, cephalopods, and verte-brates), insects have seemed to possess only the short-chainenzyme. We have now also characterized a medium-chainalcohol dehydrogenase in Drosophila. The enzyme is identicalto insect octanol dehydrogenase. It Is a typical classm alcoholdehydrogenase, similar to the correspondin human form(70% residue identity), with mostly the same residues involvedin substrate and coenzyme interactions. Changes that do occurare conservative, but Phe-Si is offunctional interest in relationto decreased coenzyme binding and Increased overall activity.Extra residues versus the human enzyme near position 250affect the coenzyme-binding domain. Enzymatic properties aresimilar-i.e., very low activity toward ethanol (K. beyondmeasurement) and high selectivity for formaldehyde/glu-tathione (S-hydroxymethylgutathione; kt/Km = 160,000min'1 mM'1). Between the present class m1 and the ethanol-active class I enzymes, however, patterns of variability differgreatly, highlighting fundamentally separate molecular prop-erties of these two alcohol dehydrogenases, with class Imresembling enzymes in general and class I showing highvariation. The gene coding for the Drosophila class HI enzymeproduces an mRNA of about 1.36 kb that is present at alldevelopmental stages of the fly, compatible with the constitu-tive nature of the vertebrate enzyme. Taken together, theresults bridge a previously apparent gap in the distribution ofmedium-chain alcohol dehydrogenases and establish a strictlyconserved classm enzyme, consistent with an important rolefor this enzyme in cellular metabolism.

The "classical" alcohol dehydrogenase is part of a wide-spread system of zinc-containing enzymes (1). In mammaliantissues, at least six classes of this enzyme occur. They differconsiderably and represent stages between separate enzymesand ordinary isozymes. Class I is the well-known liverenzyme with ethanol dehydrogenase activity (2), class III isidentical with glutathione-dependent formaldehyde dehydro-genase (3), class IV is a form preferentially expressed instomach (4, 5), while classes II, V, and VI, although littlestudied, are known also to exhibit distinct properties (6, 7,44). The class origins have been traced to gene duplicationsearly in vertebrate evolution [the I/III duplication (8)] orduring that evolution [the IV/I duplication (5)], with emerg-ing activities toward ethanol (9); class III corresponds to anancestral form. These properties and the different evolution-

ary patterns, with class III being "constant" and class I"variable" (10), result in a consistent picture of the enzymesystem and place the classes of medium-chain alcohol dehy-drogenases as separate enzymes in the cellular metabolism.

Similarly, another protein family, short-chain dehydroge-nases, has also evolved into a family comprising manydifferent enzyme activities, including an alcohol dehydroge-nase (11). This form operates by means of a completelydifferent catalytic mechanism and is related to mammalianprostaglandin dehydrogenases/carbonyl reductase (12).Thus far, this alcohol dehydrogenase has been found ininsects, the Drosophila enzyme being recognized early todiffer from the zinc-containing alcohol dehydrogenases (13,14). Its properties in various Drosophila species are wellestablished (15).These two alcohol dehydrogenase types demonstrate that

ethanol dehydrogenase activity has evolved in different man-ners, with many organisms now employing a medium-chainenzyme, while others depend on a short-chain enzyme. Themedium-chain family has not been identified in insects,although it is of ancient origin and has been characterized inother eukaryotes and in prokaryotes. We now show that thefamily is indeed present also in insects and that its majorrepresentative is the typical class III type. In fact, the enzymeturns out to be identical to Drosophila octanol dehydroge-nase, long known (16-20) but little studied. We have char-acterized the enzyme from Drosophila melanogaster enzy-matically and structurally to prove its consistency with otherclass III forms. We also have identified and sequenced thecorresponding gene§ and detected its transcription product atall developmental stages. Thus, the family is now known tobe present essentially in all life forms, supporting the viewthat medium-chain alcohol dehydrogenases are universalfactors in cellular defense mechanisms from prokaryotes tohumans. In addition, we find fundamental differences be-tween the class I and III enzymes, defining separate prop-erties of these related proteins.

MATERIALS AND METHODSProtein. D. melanogaster whole flies were bred and har-

vested as described (21). After homogenization, centrifuga-tion, isoelectric focusing, and activity staining with 1 mMoctanol or 33 mM ethanol at pH 10 and 1 mM glutathione/1mM formaldehyde atpH 8, alcohol dehydrogenase activity ofthe medium-chain type was detected and purified by utilizingion-exchange chromatography on DEAE-Sepharose, affinitychromatography on AMP-Sepharose, and a fast protein liquidchromatography (FPLC) step on Mono Q as described for theclass III enzyme from other sources (9). Short-chain alcoholdehydrogenase activity was also monitored (isopropanol at

§The sequence reported in this paper has been deposited in theGenBank data base (accession no. U07641).

4980

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

9, 2

021

Proc. NatL Acad. Sci. USA 91 (1994) 4981

pH 8.6), pooled after the ion-exchange chromatography step,and purified to homogeneity on Blue-Sepharose (22). Thestrain used contained the adhF allele yielding the rapidlymigrating form of the short-chain enzyme.

Structural Analysis. The pure protein was carboxymethy-lated by treatment with 14C-labeled iodoacetate and digestedin separate batches with proteolytic enzymes (8). Peptidedigests were fractionated by reverse-phase HPLC, and allfragments obtained were submitted to structural analysis.Amino acid compositions were determined with a PharmaciaLKB Alpha plus analyzer after acid hydrolysis for 24 hr at1100C with 6M HCl/0.5% phenol, and sequence degradationswere carried out with MilliGen Prosequencers 6600 and 6625utilizing arylamine coupling for membrane attachments orwith an Applied Biosystems 470 sequencer with an on-line120A analyzer.PCR Amplfication and Northern Analysis. Peptide struc-

tures determined were utilized for construction oftwo 23-merdegenerate oligonucleotide probes (corresponding to aminoacid residues 92-99 and 261-268), which served as primers forPCR amplification with genomic DNA of several Drosophilaspecies. Total cellular RNA was isolated by the guanidiniumisothiocyanate method (23) from larval, pupal, and adult D.melanogaster; separated by 1.2% agarose/formaldehyde gelelectrophoresis; and transferred to a nylon membrane (Am-ersham) for hybridization at high stringency (42TC in 50%formamide) (24). A control rehybridization was performedwith a D. melanogaster actin gene probe. Autoradiographswere measured with an UltroScan XL (Pharmacia LKB)enhanced laser densitometer.Enzymatic Characterization. Substrate specificities were

screened by activity staining with ethanol, isopropanol, oc-tanol, and S-hydroxymethylglutathione (formed by sponta-neous reaction of formaldehyde and glutathione) (9). Km andkin values were determined with alcohols at pH 10.0 and withS-hydroxymethylglutathione at pH 8.0 (5).

Structural Comparisons. The structure obtained was cor-related with the three-dimensional model deduced for humanclass III alcohol dehydrogenase (25) to evaluate all replace-ments. The conformational representation in Fig. 4 wasprepared by using a program supplied by Protein Science (26)and the coordinates (27) in the Protein Data Bank (28, 29) ofthe related (25) class I human alcohol dehydrogenase. Align-ments required only few insertions and utilized the class IIIalcohol dehydrogenases from a prokaryote (30), yeasts (31,32), a cephalopod (33), and vertebrates (34).

RESULTSPresence of Class II Alcohol Dehydrogenase (Octanol De-

hydrogenase). Homogenates of D. melanogaster were sub-mitted to isoelectric focusing and subsequent activity stainingwith ethanol, isopropanol, octanol, and formaldehyde/glutathione. Results (Fig. 1) clearly show the presence of twoenzyme types with partly overlapping substrate specificities.One, active with ethanol, isopropanol, and octanol, repre-sents the well-known Drosophila short-chain alcohol dehy-drogenase (13-15), which is known in multiple electropho-retic forms (35), and confirms that this is the only ethanol-active alcohol dehydrogenase in Drosophila. The other isactive with octanol and formaldehyde/glutathione in a man-ner typical of mammalian medium-chain class III alcoholdehydrogenase. This suggests that the zinc-containing classIII enzyme is present in Drosophila and that this glutathione-dependent formaldehyde dehydrogenase may represent thelittle-studied Drosophila octanol dehydrogenase previouslyreported (16-20) but not characterized structurally.The glutathione-dependent formaldehyde dehydrogenase

was purified 1900-fold in a 52% yield, resulting in a homo-geneous preparation (Fig. 1, lanes 5 and 8) after chromatog-

A : iB

t~~~~~.a1,-f 40. Z

FIG. 1. Gel electrophoretic patterns of homogenates (A) andpurified alcohol dehydrogenases (B and C) of D. melanogaster. (A)Isoelectric focusing under native conditions and activity stainingwith formaldehyde/glutathione (lane 1), octanol (lane 2), isopropanol(lane 3), and ethanol (lane 4). (B) Isoelectric focusing as inA, but withprotein staining ofthe purified proteins, octanol dehydrogenase (lane5), and short-chain dehydrogenase (lane 6). (C) Isoelectric focusingafter SDS/polyacrylamide gel electrophoresis and protein staining ofstandard proteins (lane 7; molecular masses 94, 67, 43, 30, 20.1, and14.4 kDa from top to bottom) and pure octanol dehydrogenase (lane8). In lanes 3, 4, and 6, a minor more-acidic form is visible,representing one ofinterconvertible forms in flies homozygous at theADH locus (35).

raphy on DEAE-Sepharose, AMP-Sepharose, and Mono QFPLC column in a protocol similar to that for other class Illalcohol dehydrogenases (9). All octanol dehydrogenase ac-tivity was monitored and coincided either with that of theglutathione-dependent formaldehyde dehydrogenase throughall purification steps or with the short-chain alcohol dehy-drogenase, which was also purified. The pure glutathione-dependent formaldehyde dehydrogenase exhibited both ac-tivities, with values (Table 1) typical of class Ill alcoholdehydrogenase and patterns similar to those of the corre-sponding human enzyme (36-38). The specific activity ob-tained was 12 units/mg with glutathione/formaldehyde,while the activity toward 1 mM octanol was 11 units/mg.Chromatography on Superose-12 indicated a molecular massof about 80 kDa, compatible with a dimer of40-kDa subunitsand in agreement with the estimate from SDS/polyacryl-amide gel electrophoresis (Fig. 1C). Combined with thestructural analysis (below), all results now show that Dro-sophila octanol dehydrogenase is a class III medium-chainalcohol dehydrogenase and establish the conserved catalyticactivity of this enzyme.

Table 1. Enzymatic properties of D. melanogaster medium-chainclass III alcohol dehydrogenase compared to those of human classIII alcohol dehydrogenase

Km, kCat, kct/Km,Substrate mM mni1 min-lmM-

Ethanol NS(NS)

Pentanol 5.4 840 160(22) (240) (11)

Octanol 0.51 1,300 2,500(1.2) (220) (180)

12-Hydroxydodecanoate 0.04 840 21,000(0.060) (170) (2,800)

NAD+(alc) 0.13(0.070)

S-Hydroxymethyl-GSH 0.006 960 160,000(0.004) (200) (50,000)

Values for the Drosophila enzyme experimentally determined atpH 10 in 0.1 M glycine/NaOH for the alcohol substrates and at pH8 in 0.1 M sodium pyrophosphate with 2.4 mM NAD+ for S-hy-droxymethylglutathione. Values for the human enzyme from refs.36-38 are given below, within parentheses for comparison. NS, notsaturable. NAD+(alc) indicates values in the alcohol dehydrogenasereaction measured with 0.5 mM octanol (pH 10).

Biochemistry: Danielsson et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

9, 2

021

4982 Biochemistry: Danielsson et al.

(1) (10) (20) (30) (40) (50) (60) (70) (80)Human ANE K G S E I A Y C I KDrosophila SATEGKVITCKAAVAWEAKKPLVIEDIEVAPPKAHEVRIKITATGVCHTDAFTLSGADPEGLFPVVLGHEGAGIVESVGEGVTN

1 l0 20 30 40 50 60 70 80

(90) (l00) (100) (120) (130) (140) (150) (160) (170) (080)L T P G LNP V K L D FT KTIL Y S VA DPL DFKAGDHVIALYIPQCNECKFCKSGKTNLCQKIRLTQGAGVMPEGTSRLSCKGQQLFHFMGTSTFAEYTVVADISLTKINEKAPLEKVCLLGCGISTGY

90 l00 100 120 130 140 150 160 170 180

(190) (200) (210) (220) (230) (240) (250) (260) (270)V L P V F G IM V SR I V K AR E A ECI Q FSK--P EV EM V S KV

GAALNTAKVEAGSTCAVWGLGAVGLAVGLGCKKAGAGKIYGIDINPDKFELAKKFGFTDFVNPKDVADKGSIQNYLIDLTDGGFDYTFECIGNVNTMR090 200 210 220 230 240 250 260 270 280

(280) (290) (300) (310) (320) (330) (340) (350) (360) (370)A C V V AS E A T T T K ES SE MS KIK V N SFDE K E S K TVV ISALEATHKGWGTSVVIGVAGAGQEISTRPFQLVVGRVWKGSAFGGWRSVSDVPKLVEDYLKKDLLVDEFITHELPLSQINEAFDLMHKGESIRSIIKY

290 300 310 320 330 340 350 360 370

FIG. 2. Primary structure ofD. melanogaster class III alcohol dehydrogenase (lower, continuous sequence) and its relationship to the humanclass HI structure (upper sequence; residues cited only where different; in addition, the human enzyme lacks residues corresponding to the threefirst residues of the Drosophila form). Peptides passed by Edman degradations are denoted by lines beneath the sequences and were obtainedby cleavages with a lysine-specific protease (upper lines) and with a Glu-specific protease (lower lines). Positional numbers (within parenthesis)above the human sequence line refer to the human class I enzyme and are given to allow correlation with the functional residues, which aregenerally known under their numbers in the class I enzymes, while numbers below the Drosophila sequence refer to the Drosophila class menzyme now determined. Initiator methionine is not included since the protein is N-terminally blocked and is concluded to be acetylated likethe human form.

Structure, Presence in Different Species, and Expression atAU Developmental Stages. The enzyme was carboxymethy-lated and digested in different batches with glutamic acid- andlysine-specific proteases, respectively. Each digest was frac-tionated by reverse-phase HPLC, and peptides were submit-ted to sequence analysis. These results, combined with DNAdata (below), gave the primary structure of the protein chain(Fig. 2). Only the N-terminally blocked peptides were notdegraded, and positions 1-3 rely on the DNA data.

Oligonucleotides (corresponding to positions 92-99 and261-268) were synthesized and used for PCR amplificationswith DNA from five Drosophila species belonging to threedifferent subgenera. Data obtained (Fig. 3A) show that thegene coding for the class III enzyme is present in all of thespecies tested. However, slightly different sizes of the am-plified fragments suggest that there is some variability in thegenomic region flanked by the two PCR primers.

AN. DiDI)I 1)1 IDs rIm N

B

I P .A

FIG. 3. Presence of a class Ill alcohol dehydrogenase/octanoldehydrogenase gene in several Drosophila species (A) and expres-sion in different developmental stages (B). (A) PCR amplificationwith DNA of five Drosophila species: D. immigrans (lane Di); D.hydei (lane Dh); D. lebanonensis (lane Dl); D. subobscura (lane Ds);and D. melanogaster (lane Dm). Lane M shows Hae III-digestedpBR322 as molecular weight markers. Amplification was performedin 100 Ad containing 1 pg of genomic DNA, 40 pmols of eachdegenerate primer, 1.5 units of Taq polymerase (Promega) in 2.5 mMMgCl2, and 200 pM each dNTP. After 2 min at 94CC and 45 cyclesof 940C for 60 s, 50TC for 90 s, and 72TC for 90 s, 15 Al was resolvedon 1.2% agarose gels in Tris borate/EDTA buffer and stained withethidium bromide. Units are in kb. (B) Northern analysis with larval(lane L), pupal (lane P), and adult (lane A) total cellular RNA fromD. melanogaster. Units are in kb.

Using the PCR-amplified products, we cloned and se-quenced the D. melanogaster gene, confirming the proteinstructure. Total RNA from larval, pupal, and adult D. mel-anogaster was also used for Northern analysis with thePCR-amplified product (Fig. 3B; an actin gene probe was alsoused). A single band ofcomparable intensity (as measured bylaser densitrometry with the actin control) and a1.36 kb wasdetected in each sample. This indicates that expression oftheclass III gene is not confined to a particular developmentalstage but appears to be abundant during the life span of theorganism. Together, all data support a wide distribution ofthe enzyme in the Drosophila genus. The constant presenceof the transcript at all developmental stages is compatiblewith a constitutive pattern of expression.

DISCUSSIONIdentification of Medium-Chain Alohol Dehydrogese as

Octanol Dehydrogenase in Drosophila. The demonstration ofa medium-chain alcohol dehydrogenase in Drosophila estab-lishes that this protein family occurs throughout the livingworld. It has previously been characterized structurally inprokaryotes (30) and in many highly different eukaryotic lines(31-34) but thus far not in Drosophila, where the short-chainalcohol dehydrogenase appeared to constitute a peculiarfeature of alcohol oxidation. Nevertheless, the short-chainenzyme is the only one with appreciable ethanol dehydroge-nase activity in Drosophila (Fig. 1), even though the medium-chain family is present, as is now evident. Its class III formcoexists with the short-chain enzyme and has both unalteredenzymatic properties and a structure conserved in relation tothe human enzyme. The absence of a class I medium-chainalcohol dehydrogenase in Drosophila apparently reflects thelater enzymogenesis of that enzyme type (9) but does notimply that the protein family as such or the class III ancestralform is absent.The extremely wide occurrence ofthis protein highlights its

general importance and suggests that it has a role in basiccellular metabolism. Moreover, the present results identifyoctanol dehydrogenase, which has been discussed previously(16-20) but with somewhat contradictory estimates regardingmolecular weight and quaternary structure, as being identicalto the class III medium-chain alcohol dehydrogenase. Thus,the octanol dehydrogenase is involved also in glutathione-dependent elimination of formaldehyde. However, negativemutants are viable (32, 39), suggesting further multiplicityand the existence of a back-up system.

Proc. Natl. Acad Sci. USA 91 (1994)

Dow

nloa

ded

by g

uest

on

Mar

ch 2

9, 2

021

Proc. NatL Acad. Sci. USA 91 (1994) 4983

Correlation with Functional Properties. The identificationand analysis of Drosophila class III alcohol dehydrogenasemeans that five divergent forms (conserved residues, 46%6) ofclass III alcohol dehydrogenase have now been characterizedstructurally-mammalian forms including the human enzyme(34), a cephalopod enzyme (33), a Drosophila enzyme (thiswork), and two yeast enzymes (31, 32). Their properties canbe compared with those of the classical liver alcohol dehy-drogenase ofclass I, whose structure, within the same family,has been analyzed recently to a level showing approximatelythe same residue divergence (conserved residues, 42%) byanalysis of five major vertebrate lines (40).The conservation of functionally important residues in

class III is extensive. Of 35 positions participating in coen-zyme or substrate interactions in the class I enzyme (41) andconsidered to do so also in the class III enzyme (25, 42), noless than 28 are strictly conserved between the human andDrosophila class III enzymes, and all but 1 of the 7 ex-changed constitute just minor variations encountered also inother species. Position 51 is the single exception. There, theclass III Drosophila enzyme has a phenylalanine residue, asdoes the Escherichia coli enzyme (30), while human and theother class III forms have a tyrosine residue [and the wholefamily has a histidine, tyrosine, or serine residue (5, 33, 40)].This residue usually participates in hydrogen bonding withthe ribose moiety of the NMN part of the coenzyme (25).Although the hydrogen bond may have little effect (43),Phe-51 cannot donate such a bond to the coenzyme. There-fore, this exchange may contribute an explanation to the

V2M

IIIl

HumanOctopusDrosophilaCandida

150

weakened coenzyme binding of the Drosophila enzyme and,hence, to its increased activity relative to the human enzyme(Table 1).

Disregarding the functionally important residues, the Dro-sophila enzyme has two extra residues in relation to thehuman class III form. These extra residues, close to position250 (Lys-251 and Gly-252 in Fig. 2, or adjacent residues,depending on the alignment chosen), are also present in theyeast enzyme (31, 32) but not in the octopus enzyme (33).This region corresponds to a surface helix in the coenzymebinding domain (Fig. 4 Upper Left), and it appears possibleto extend the helix without adverse consequences.The conservation of class III stands in marked contrast to

the spread within the class I enzyme. Just among vertebrates,class I enzyme residue conservation [55% for the human/fishpair (42)] is lower than that for class III across separateeukaryotes (63% for the human/yeast pair). Among the 35functionally important positions, no fewer than 20 vary,many to a great extent (40, 42). Glycolytic enzymes-e.g.,glyceraldehyde-3-phosphate dehydrogenase (human/yeast,63-65% residue identity, depending on the types compared)and enolase (human/yeast, 61-64% identity)-demonstratethat the class III alcohol dehydrogenase variation (human/yeast, 63%) is "normal" for an enzyme of this type in a fixedmetabolic pathway. It is the faster evolving class I that has an"atypical" variation. In fact, the human/yeast values for thetwo glycolytic enzymes and the class HI alcohol dehydroge-nase are surprisingly close, just within ±2%, suggestingsimilar functional restrictions for these three metabolically

50 100 150 200 250 300 350A I I I I

I 1111111 E UI EII lll 1 11 1 1 1_accham I'lYi_ "' _Y' * El*'I . dI.I 11,uu11.Saccharomyces _ _lm m l lo m s pi I po lo -,A _ ,,I,1HumanChickenAlligatorFrogCod

I

V21VIM V2M

V3,

200 250 300 350

FIG. 4. Differences in patterns of variability for classes I (bar and conformation) (Lower) and Ill (bar and conformation) (Upper). The barsgive a schematic representation ofthe residue conservations along the polypeptide chains (positions numbered), with black vertical lines denotingpositions with strictly conserved residues among the species shown. Unfilled bar areas correspond to variable segments, V1-V3I in class I andV1i1-WV2iii in class III. Their spatial positions are shown with thick lines inside the models of the class I and III enzymes (25, 27). Theconformation is that from the Protein Data Bank coordinates (28, 29) of the human class I enzyme (27) when using a program supplied by ProteinScience (26).

. - ... I-.. II.$ I i. I.

im m . m6 ohmm mb A sm ' -. im . ohm i mm. '. its . . mhm. ..' . -. t -iI| |- He And - M l| ]

Biochemistry: Danielsson et al.

I

VIi

50 160

Dow

nloa

ded

by g

uest

on

Mar

ch 2

9, 2

021

4984 Biochemistry: Danielsson et al.

fixed enzymes. This agreement adds strength to the conclu-sion of significantly different natures of the class I and IIIalcohol dehydrogenases.Fundamental Differences in Molecular Architecture of Re-

lated Proteins. Apart from the differences in overall conser-vation, the actual distributions of the constant and variablesegments of the class I and III alcohol dehydrogenases arefundamentally different. In class I, three segments (V1-V30)stand out as variable (empty spaces in the bottom bar in Fig.4) and have been noted to make class I peculiar amongproteins in general by suggesting hypervariability in impor-tant segments (40). As shown in the conformation (Fig. 4Lower), they correspond to a segment adjacent to the activesite (V1i), a part of the loop around the second zinc atom(V20), and a part of the subunit-subunit interacting segment(V30). These three segments all are concentrated on one sideof the molecule (toward the observer in Fig. 4). Therefore,apart from affecting the functional areas, their covariabilitymay have even further implications, suggesting variability ofa particular side of the class I molecule.

In contrast, these segments are not at all variable in classIII but are exactly those much conserved (bar in Fig. 4Upper), supporting the view that class III represents a"normal" enzyme, with maximal conservation at the activesite and other important segments. Nevertheless, class Illalso has segments of variability, but its two such segments(V1m and V2E1) affect nonfunctional, superficial regions,completely differently positioned regarding both functionalrepresentation and molecular surfaces (Fig. 4 Upper). Inter-estingly, though, V2I corresponds to the helix constitutingone of the two domain-interconnections of the subunit (Fig.4). Since domain movements are known to be associated withcoenzyme binding in the class I enzyme (41), this segmentmay indicate further functional differences in catalyticallyactive states between the classes.The remaining classes are not yet known in similar detail.

Nevertheless, their inclusion in bar comparisons of the typein Fig. 4 (as given in ref. 40) suggests that the class I patternis typical of variable classes of medium-chain alcohol dehy-drogenases.

In conclusion, we notice that fundamentally different in-ternal variability patterns affect two related proteins of aprotein family. These patterns add to the overall differencesbetween the classes and show that differences apply tobuilding elements in the molecular architecture of the pro-teins. Class III behaves as enzymes in general, constant infunction, enzymology, overall structure, and important seg-ments, while class I is emerging in function, exhibitingenzymatic differences, rapid evolutionary changes, and van-ability at important regions. The two variability patterns (Fig.4) illustrate fundamental differences between an evolving anda constant protein within a single family, and correlate thedifferences with the molecular architecture and enzymaticproperties.

We are grateful to Dr. Bengt Persson (Department of MedicalBiochemistry and Biophysics) for supplying the program utilized forthe bar constructions in Fig. 4, and to Heldne Olsson (Center forBiotechnology) for assistance with structural analysis. This studywas supported by the Swedish Medical Research Council (Project03X-3542), the Swedish Alcohol Research Fund (Project 88/12:4),and a Spanish Comisi6n Interministerial de Ciencia y Tecnologfagrant (BI092-0591-C02-02).

1. Jdmvall, H., Danielsson, 0., Eklund, H., Hjelmqvist, L., H66g, J.-O.,Par6s, X. & Shafqat, J. (1993) in Enzymology and Molecular Biology ofCarbonyl Compounds 4, eds. Weiner, H., Crabb, D. W. & Flynn, T. G.(Plenum, New York), pp. 533-544.

2. Vallee, B. L. & Bazzone, T. J. (1983) Curr. Top. Biol. Med. Res. 8,219-244.

3. Koivusalo, M., Baumann, M. & Uotila, L. (1989) FEBS Lett. 257,105-109.

4. Moreno, A. & Pares, X. (1991) J. Biol. Chem. 266, 1128-1133.5. Pares, X., Cederlund, E., Moreno, A., Hjelmqvist, L., Farr6s, J. &

JOrnvall, H. (1994) Proc. Nat!. Acad. Sci. USA 91, 1893-1897.6. Jmrnvall, H., H66g, J.-O., von Bahr-Lindstr6m, H. & Vallee, B. L.

(1987) Proc. Nat!. Acad. Sci. USA 84, 2580-2584.7. Yasunami, M., Chen, C.-S. & Yoshida, A. (1991) Proc. Natl. Acad. Sci.

USA 88, 7610-7614.8. Cederlund, E., Peralba, J. M., Pares, X. & J6rnvall, H. (1991) Biochem-

istry 31, 2811-2816.9. Danielsson, 0. & J6rnvall, H. (1992) Proc. Natl. Acad. Sci. USA 89,

9247-9251.10. Yin, S.-J., Vagelopoulos, N., Wang, S.-L. & J6rnvall, H. (1991) FEBS

Lett. 283, 100-103.11. Persson, B., Krook, M. & J6rnvall, H. (1991) Eur. J. Biochem. 261,

537-543.12. Krook, M., Ghosh, D., Str6mberg, R., Carlquist, M. & J6mvall, H.

(1993) Proc. Natl. Acad. Sci. USA 9W, 502-506.13. Schwartz, M. F. & J6rnvall, H. (1976) Eur. J. Beochem. 68, 159-168.14. Thatcher, D. R. & Sawyer, L. (1980) Biochem. J. 187, 884-886.15. Atrian, S., Marfany, G., Albalat, R. & GonzIez-Duarte, R. (1992)

Biochem. Genet. 11, 19-29.16. Urspnrng, H. & Leone, J. (1965) J. Exp. Zool. 161, 147-154.17. Sieber, F., Fox, D. J. & Ursprung, H. (1972) FEBS Lett. 26, 274-276.18. Dickinson, W. J. & Sullivan, D. T. (1975) Gene-Enzyme Systems in

Drosophila (Springer, Heidelberg), pp. 78-80.19. Madhavan, K., Conscience-Egli, M., Sieber, F. & Ursprung, H. (1973)

J. Insect Physiol. 19, 235-241.20. Ogonji, G. 0. (1971) J. Exp. Zool. 178, 513-522.21. Ribas de Pouplana, L., Atrian, S., Gonzilez-Duarte, R., Fothergill-

Gilmore, L. A., Kelly, S. M. & Price, N. C. (1991) Biochem. J. 276,433-438.

22. Juan, E. & GonzAlez-Duarte, R. (1980) Biochem. J. 189, 105-110.23. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159.24. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning:A

Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY), 1stEd.

25. Eklund, H., Miller-Wille, P., Horales, E., Futer, O., Holmquist, B.,Vallee, B. L., H66g, J.-O., Kaiser, R. & J6rnvall, H. (1990) Eur. J.Biochem. 193, 303-310.

26. Richardson, D. C. & Richardson, J. S. (1992) Protein Sci. 1, 3-9.27. Hurley, T. D., Bosron, W. F., Hamilton, J. A. & Amzel, L. M. (1991)

Proc. Natl. Acad. Sci. USA 88, 8149-8153.28. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr.,

Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. & Tasumi,M. (1977) J. Mol. Biol. 112, 535-542.

29. Abola, E. E., Bernstein, F. C., Bryant, S. H., Koetzle, T. F. & Weng,J. (1987) in Crystallographic Databases-Information Content, SoftwareSystems, Scientific Applications, eds. Allen, F. H., Bergerhoff, G. &Sievers, R. (Data Commission Intl. Union Crystallogr., Bonn), pp.107-132.

30. Gutheil, W. G., Holmquist, B. & Vallee, B. L. (1992) Biochemistry 31,475-481.

31. Sasnauskas, K., Jomantiene, R., Janu~ka, A., Lebediene, E., Lebedys,J. & Janulaitis, A. (1992) Gene 122, 207-211.

32. Wehner, E. P., Rao, E. & Brendel, M. (1993) Mol. Gen. Genet. 237,351-358.

33. Kaiser, R., Fernindez, M. R., Pards, X. &J6rnvall, H. (1993)Proc. Natl.Acad. Sci. USA ", 11222-11226.

34. Kaiser, R., Holmquist, B., Vallee, B. L. & Jmrnvall, H. (1989) Biochem-istry 28, 8432-8438.

35. Jacobson, K. B., Murphy, J. B., Knopp, J. A. &Ortiz, J. R. (1972)Arch.Biochem. Biophys. 149, 22-35.

36. Wagner, F. W., Pares, X., Holmquist, B. & Vallee, B. L. (1984) Bio-chemistry 23, 2193-2199.

37. Moulis, J.-M., Holmquist, B. & Vallee, B. L. (1991) Biochemistry 30,5743-5749.

38. Uotila, L. & Koivusalo, M. (1974)J. Biol. Chem. 249, 7653-7663.39. Voelker, R. A., Langley, C. H., Leigh-Brown, A. J., Ohnishi, S., Dick-

son, B., Montgomery, E. & Smith, S. C. (1980) Proc. Nat!. Acad. Sci.USA 77, 1091-1095.

40. Persson, B., Bergman, T., Keung, W. M., Waldenstr6m, U., Holmquist,B., Vallee, B. L. & J6rmvall, H. (1993) Eur. J. Biochem. 216, 49-56.

41. Eklund, H., Samama, J.-P. & Jones, T. A. (1984) Biochemistry 23,5982-5996.

42. Danielsson, O., Eklund, H. & JMrnvall, H. (1992) Biochemistry 31,3751-3759.

43. Schmid, F., Hinz, H.-J. & Jaenicke, R. (1978) FEBS Lett. 87, 80-82.44. Zheng, Y.-W., Bey, M., Liu, H. & Felder, M. R. (1993)J. Biol. Chem.

268, 24933-24939.

Proc. Nad. Acad Sci. USA 91 (1994)

Dow

nloa

ded

by g

uest

on

Mar

ch 2

9, 2

021