Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis

Ž .Biochimica et Biophysica Acta 1339 1997 133–142

Structural and biochemical studies of alcohol dehydrogenase isozymesfrom KluyÕeromyces lactis

Argante Bozzi a, Michele Saliola b, Claudio Falcone b, Francesco Bossa c,), Filippo Martini d

a Department of Technological and Biomedical Sciences, UniÕersity of L’Aquila, I-67100 Aquila, Italyb Pasteur Institute-Cenci Bolognetti Foundation, Department of Cell and DeÕelopmental Biology UniÕersity of Rome ‘La Sapienza’,

I-00185 Rome, Italyc Department of Biochemical Sciences ‘ A. Rossi Fanelli’ UniÕersity of Rome ‘La Sapienza’ and CNR Center for Molecular Biology, P.le

A. Moro, 5, I-00185 Rome, Italyd Institute of Biochemical Sciences UniÕersity ‘G. D’Annunzio’ of Chieti, I-66100 Chieti, Italy

Received 9 September 1996; revised 3 December 1996; accepted 5 December 1996

Abstract

Ž .The cytosolic and mitochondrial alcohol dehydrogenases from KluyÕeromyces lactis KlADHs were purified andcharacterised. Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependentand exhibited catalytic properties similar to those previously reported for other yeast ADHs. Conversely, the mitochondrialisozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at anincreased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol.Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources,were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP. Since Asp-223 ofhorse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADHisozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to theother residues lining the coenzyme binding pocket of the mitochondrial isozymes. The relatively low specificity of themitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibilityof the corresponding catalytic pockets. An involvement of the mitochondrial KlADH isozymes in the physiologicalreoxidation of the cytosolic NADPH was also hypothesized. Moreover, both cytosolic and KlADH IV isozymes have anadditional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of2-mercaptoethanol.

Ž . Ž . Ž .Keywords: Alcohol dehydrogenase; Isozyme; NAD P ; Mitochondrion ; K. lactis

Abbreviations: ADH, alcohol dehydrogenase; SADH, KlADH,HLADH, alcohol dehydrogenases from Saccharomyces cere-Õisiae, KluyÕeromyes lactis and horse liver; SDS-PAGE, sodiumdodecyl sulfate polyacrylamide gel electrophoresis; alcohol dehy-

Ž .drogenase EC 1.1.1.1) Corresponding author. Fax: q39 6 4440062; E-mail:

[email protected]

1. Introduction

Ž .Alcohol dehydrogenases ADHs from a wide vari-w xety of sources have been extensively studied 1 .

Almost all the cytosolic ADHs so far characterisedhave been grouped in at least five classes, whereas

0167-4838r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0167-4838 96 00225-7

( )A. Bozzi et al.rBiochimica et Biophysica Acta 1339 1997 133–142134

much less information is available on the mitochon-drial isozymes. Mitochondrial ADHs have been found

Žin some yeast species Saccharomyces cereÕisiae and) w xKluyÕeromyces lactis 2–4 . In S. cereÕisiae two

cytosolic ADHs have been identified, SADH I whichis the fermentative isozyme involved in the reduction

w xof acetaldehyde to ethanol 2 and SADH II whichcatalyses the oxidation of ethanol to acetaldehyde.The gene encoding the latter activity is repressed atthe transcriptional level in the presence of glucoseand de-repressed when the sugar concentration de-

w xcreases in the growth medium 5,6 . The mitochon-drial SADH III activity seems to play an oxidative

w xfunction not relevant for the yeast cell 7 . AnotherADH gene, namely SADH 4, is present in S. cere-Õisiae; the encoded protein is not related to theprevious ones but shows some similarity to an iron-activated bacterial alcohol dehydrogenase from Zy-

w xmomonas mobilis 8 . Recently, during the sequenc-ing project of the S. cereÕisiae genome, another

Ž . Ž .ADH gene SADH 5 highly homologous )80% toSADH 1, SADH 2 and SADH 3 has been found on

w xchromosome II 9 .The alcohol dehydrogenase system of the yeast K.

lactis includes four structural genes, encoding twoŽ .cytosolic KlADH I and KlADH II and two mito-Ž .chondrial KlADH III and KlADH IV isozymes

w x3,4,10 . Both cytosolic activities seem to play, simi-larly to SADH I, a fermentative role and the corre-sponding genes are essentially constitutive and pref-

w xerentially expressed in glucose grown cells 3,4,11 .On the contrary, the genes encoding the mitochon-drial isozymes are tightly regulated by the carbonsource; in fact, KlADH 4 is specifically induced byethanol at the transcriptional level and is not sensitive

w xto glucose repression 12 , while KlADH 3 is inducedby different respiratory carbon sources except ethanolw x11 . At present, the physiological function of the twomitochondrial isozymes is not clear.

K. lactis mutant strains carrying only one of thefour ADH genes can still grow on ethanol, although

w xat different rates 13 . The genes encoding the mito-chondrial and cytosolic ADH isozymes from both S.

w x w xcereÕisiae 7,14,15 and K. lactis 3,4,10 have beenidentified and sequenced. All the deduced ADHamino-acid sequences from K. lactis and S. cere-

Ž .Õisiae show a high degree of identity 75–90% .Sequence differences have been observed mainly in

the mitochondrial-targeting presequences as well asw xin the C-terminal regions 4,10 .

Most yeast ADHs bind two zinc ions, one withcatalytic function, located at the active-site, and the

w xother with structural function 16 . Only three out ofthe seven zinc ligands in horse liver ADH, namelyCys-46, His-67 and Cys-174, have been found to beinvariant among the ADH sequences from various

w xsources 17,18 . Moreover, thiol compounds, such as2-mercaptoethanol, were able to inhibit selectivelythe alcohol dehydrogenase from horse liver, but notthe commercial yeast ADH, probably by interfering

Ž .with the protein cysteine residue s responsible forw xbinding the catalytic zinc ion 19 .

It has also been reported that yeast ADHs from S.cereÕisiae have more restricted substrates specificity

w xthan mammalian liver isozyme 20 . The substratespecificity of SADH I was decreased by site-directedmutagenesis modification of some active-site amino-

w xacid residues 21 .Almost all alcohol dehydrogenases from different

sources use NADq rather than NADPq as a coen-zyme. The three-dimensional structure of horse liverADH has suggested that Asp-223 is important in

w xdetermining this specificity 16 . Moreover, Asp-223is highly conserved in all the NAD-dependent ADHsw x17,18 and its replacement by Gly in SADH I allowsthe mutant enzyme to accept NADq and NADPq

w xequally well 22 .The presence of four ADH genes in K. lactis was

in some way surprising since this organism, differ-ently from S. cereÕisiae, shows a predominant respi-ratory metabolism and does not accumulate highethanol levels during growth on glucose containing

Ž w x.medium for a review see Ref. 23 .Despite a high sequence conservation, the differ-

ence in the number of ADH genes and their regula-tion by carbon source between S. cereÕisiae and K.lactis suggests that the corresponding activities aredue to peculiar biochemical properties and play dif-ferent physiological roles in the two organisms.

To get further information on the specific bio-chemical properties of the alcohol dehydrogenasesfrom K. lactis, we undertook a study on the purifica-tion and biochemical characterization of theseisozymes and compared the results with those ob-

w xtained from ADHs from S. cereÕisiae 24 and othersources.

( )A. Bozzi et al.rBiochimica et Biophysica Acta 1339 1997 133–142 135

2. Materials and methods

2.1. Chemicals

Ž .q Ž .NAD P and NAD P H were purchased fromŽ .Boehringer Mannheim, Germany ; alcohols and

Ž .aldehydes were from Fluka Buchs, Switzerland .Other chemicals of reagent grade were supplied from

Ž .Carlo Erba Milan, Italy .

2.2. Yeast media and cell growth

w xK. lactis CBS2359r152 25 was the wild-typestrain used in our experiments which possessed thefour alcohol dehydrogenase activities. K. lactis mu-tants harbouring single ADH activities were obtainedby gene disruption techniques and genetic crossesw x12,13 . Strains were grown aerobically at 288C in YP

Ž .medium 1% yeast extract, 1% peptone, DIFCOcontaining 2% glucose for the preparation of KlADHI and KlADH II, 2% glycerol for KlADH III and 2%

w xethanol for KlADH IV 11,13 .

2.3. Purification and assay of the KlADH isozymes

Thirty ml extract obtained from 500 ml cultures ofthe four adh mutants were subjected to the purifica-tion procedure. The purification of the KlADH I,KlADH II and KlADH III isozymes was performed

w xaccording to Scopes 26 with minor modifications,while the purification of the KlADH IV isozyme,besides some adjustment of the cited procedure, re-quired a further chromatography step. Briefly, afterthe ethanol treatment, the KlADH IV extracts wereprecipitated with 50–75% ammonium sulfate. Thepellet was resuspended in 30 mM phosphate bufferŽ .pH 7.5 and desalted by using a Sephadex G-100

Žfine column 2.5=33 cm, Pharmacia, Uppsala Swe-.den , equilibrated and eluted with the same buffer.

The active fractions were pooled and chromato-Žgraphed on a DE-52 cellulose column 1.8=13 cm,

.Whatman, England , equilibrated in 30 mM phos-Ž .phate buffer pH 7.5 and eluted with 200 ml of

0–300 mM NaCl linear gradient. After this step, theactive fractions were adjusted to 100 mM phosphatebuffer and, after the addition of 1.9 M ammoniumsulfate, were loaded onto a Phenyl-Sepharose CL-4B

Ž .column 1.3=10 cm, Pharmacia previously equili-

Ž .brated in 100 mM phosphate buffer pH 7.5 contain-ing 1.9 M ammonium sulfate. The column waswashed by decreasing to zero the concentration of theammonium sulfate and the elution of KlADH IV wasperformed isocratically with 100 mM phosphate

Ž . Ž .buffer pH 7.5 containing 20% vrv of both glyc-erol and ethanol. The active fractions were collected,concentrated and stored at y208C. Before use, bothglycerol and ethanol were removed from the KlADHIV samples by filtration on a PD-10 pre-packed

Ž .column Pharmacia equilibrated in 30 mM phos-Ž .phate buffer pH 7.5 and eluted with the same

buffer. Sometimes, the purification procedure rou-tinely used to purify the cytosolic KlADHs andKlADH III isozymes was insufficient, so that a fur-ther chromatography step on Phenyl-Sepharose CL-4B was required. In the latter case, the chromato-graphic behaviour on the hydrophobic resin was iden-

Žtical to that observed for the KlADH IV isozyme see.above .

Aliquots from each step were analysed for proteinŽ .concentration Pierce protein assay reagent and ADH

activity. The ADH activity was routinely assayed inŽ .50 mM Tris-HCl pH 8.3 in the presence of 2 mM

NADq and 100 mM ethanol in 1 ml final volume, at258C.

2.4. Structural analysis

The N-terminal amino-acid sequences of the puri-fied KlADH isozymes were determined by automatedEdman degradation using an Applied Biosystemsmodel 476A gas-phase sequencer. Protein samplesŽ .0.3–0.5 nmol were loaded onto Pro-Blott mem-

Žbranes and run on the Blott Cartridge Applied.Biosystems according to the manufacturer’s instruc-

tions.

2.5. Gel electrophoresis

Cells from 10 ml cultures were broken with glassbeads, centrifuged and the supernatants were anal-ysed by non-denaturing electrophoresis on 5% acryl-amide minigels. KlADH isozymes were revealed by

w xstaining the gels for ADH activity 2 . Electrophoreticconditions and detection of ADH activities on the gel

w xwere performed as previously described 12 .Moreover, the purity and the molecular mass of


the KlADH subunits was assessed in denaturing con-dition by SDS-PAGE on 12% precast acrylamide

Ž .minigels Bio-Rad , stained with Coomassie brilliantblue. Such analyses revealed the presence of a singleband for each isozyme preparation and the absence ofsignificant degradation during the purification proce-dures.

2.6. Molecular mass determination

The molecular mass of the KlADH III and KlADHIV monomers was calculated from the correspondinggene-derived amino-acid sequences at ExPASy WorldWide Web molecular biology server of the GenevaUniversity Hospital and the University of GenevaŽ .Switzerland . The molecular mass of native KlADHisozymes was determined on a TSK 3000 SW gel-

Žfiltration column eluted with 50 mM Tris-HCl pH.8.0 at a flow rate of 0.5 mlrmin, the effluent being

monitored at 280 nm. Molecular mass values ofnative KlADH isozymes were estimated by compari-son with a calibration mixture of standard proteins,

Ž .namely carbonic anhydrase 29 kDa , bovine serumŽ . Žalbumin 66 kDa , yeast alcohol dehydrogenase 150

. Ž . Ž .kDa , b-amylase 200 kDa and apoferritin 443 kDa .

2.7. OxidatiÕe reaction conditions

Preliminary experiments have shown that the bestbuffers for the cytosolic and mitochondrial KlADH

Ž .assays were 50 mM Hepes pH 7.3 , and 50 mMŽ .Tris-HCl pH 8.0 , respectively. Moreover, in order

to compare our results with those previously reportedw x10 , on the capability of the KlADH isozymes ofoxidising other alcoholic substrates besides ethanol,

Ž .20 mM pyrophosphate buffer pH 8.5 was also used.For the determination of the alcohol preferences ofthe KlADH isozymes, saturating concentrations ofthe different alcohols were added to the assay mix-ture which contained fixed amount of each isozyme.For the assay of KlADHs in the oxidative direction,

Ž . qthe reaction mixture 1 ml contained 2 mM NADq Ž .or NADP in 50 mM Tris-HCl pH 8.0 for mito-

Ž .chondrial isozymes, or in 50 mM Hepes pH 7.3 forthe cytosolic ones. The assay mixtures were pre-in-cubated with each isozyme for 2 min at 258C, thenthe reactions were started by adding 100 ml of 1 Malcoholic substrate. The activity was determined on a

Varian DMS 90 spectrophotometer thermostatted at258C, by monitoring the increase of absorbance at340 nm.

( )2.8. ReductiÕe fermentatiÕe reaction conditions

The fermentative reaction was performed as fol-lows: in a final volume of 1 ml, 100 ml of 3 mMNADH or NADPH, in the same buffers used for theoxidative reaction, and an appropriate amount of eachKlADH isozyme were added. Each mixture was pre-incubated 2 min at 258C, and the reaction was startedby adding 100 ml of 500 mM aldehydic substrate.The reaction was followed at 258C by monitoringspectrophotometrically the absorbance decrease at 340

Ž Ž . y1 y1.nm e NAD P Hs6.22 mM cm .340

2.9. Kinetic analysis

For the oxidative reactions of both mitochondrialand cytosolic KlADHs the concentrations of ethanoland NADq or NADPq were in the ranges 1–300mM and 0.05–20 mM, respectively, whereas for thefermentative reactions the concentrations of the alde-hydic substrates and NADH or NADPH varied in theranges 0.1–50 mM and 20–500 mM, respectively.

Enzyme concentrations used for kinetic studieswere adjusted so that absorbance changes at 340 nmwere linear for at least 2–3 min. V and K valuesmax m

were calculated from duplicate initial velocity deter-minations performed at eight–ten substrate concentra-

Ž .tions, at 258C. Catalytic efficiency K rK , whichcat mw xwas derived from V rK P E , was used for mea-max m

w xsuring the cofactor affinity of each isozyme 27 ,while the substrate preferences of the KlADHisozymes were expressed as relative reaction rates.

All fittings were carried out on a personal com-w xputer by using the ENZFITTERq program 28 , which

applies a non-linear regression data analysis. Thestandard errors of K and K values were withinm cat

10%.

2.10. ActiÕity of the KlADH isozymes in the presenceof 2-mercaptoethanol

2-Mercaptoethanol as a substrate was assayed onboth mitochondrial and cytosolic KlADHs in theconcentration range up to 300 mM in the conditions


Table 1Ž .Enzymatic properties of the ADH isozymes from K. lactis. Kinetic parameters 258C of cytosolic and mitochondrial ADHs from K.

lactis for the substrates in the fermentative and oxidative reactions

Isozyme Ethanol Acetaldehyde

K k k rK K k k rKm cat cat m m cat cat m

Cytosolic5 3 5 5KlADH I 27.0 2.5=10 9.3=10 1.2 3.6=10 3.0=104 3 4 4KlADH II 23.0 2.8=10 1.2=10 1.7 3.3=10 2.0=10

Mitochondrial4 4 5 6KlADH III 2.57 8.2=10 3.2=10 0.1 8.6=10 8.6=104 3 4 3KlADH IV 1.56 1.3=10 8.3=10 3.1 2.9=10 9.0=10

Ž .reported for the alcoholic substrates see above .Moreover, the effect of this reducing agent on theKlADH isozymes was studied after incubation for 5

Žmin with 2-mercaptoethanol concentration range. Ž .1–50 mM in 50 mM Tris-HCl pH 8.0 , at 258C

using 2 mM NADq as cofactor.

3. Results

3.1. Kinetic properties of the KlADH isozymes

The steady-state catalytic properties of the fourKlADH isozymes either towards the substrates or the

Ž .q Ž .coenzymes NAD P and NAD P H are shown inTables 1 and 2. Both KlADH I and II exhibitedK values 9- and 17-fold higher than those ofmŽethanol.

Ž .the mitochondrial isozymes Table 1 but comparablew xto that reported for S. cereÕisiae ADH I 24 , con-

firming that the cytosolic isozymes play a similar rolein ethanol production. Conversely, the K values form

ethanol of the mitochondrial KlADHs were signifi-Ž .cantly lower 5- and 8-fold than that of S. cereÕisiae

w xmitochondrial ADH III 24 . The k rK values forcat m

either ethanol or acetaldehyde of KlADH IV weresimilar to those for ethanol of the cytosolic isoformsŽ .Table 1 . On the other hand, KlADH III, showing a3.5- and 27-fold enhancement of k rK ascat mŽethanol.compared to the other three isozymes, exhibited thehighest affinity towards this substrate. KlADH IIIdisplayed also the highest value of k rKcat mŽacetaldehyde.which resulted to be 270-fold higher than that forethanol, thus revealing a marked preference of

Ž .KlADH III for acetaldehyde Table 1 .Interestingly, while cytosolic KlADHs did not ex-

Ž .hibit any interaction with NADP H the mitochon-Ž .drial isozymes worked with either NAD H or

Ž .NADP H , although the K values for the phospho-m

rylated pyridine coenzyme were always higher thanthose for the corresponding non-phosphorylated oneŽ .Table 2 . Concerning the preference of the KlADHisozymes for the pyridine coenzymes, the two mito-

Table 2Ž .Cofactor affinity of the ADH isozymes from K. lactis. Kinetic parameters 258C of cytosolic and mitochondrial ADHs from K. lactis

q qIsozyme NAD NADP NADH NADPH

K k k rK K k k rK K k k rK K k k rKm cat cat m m cat cat m m cat cat m m cat cat m

Cytosolic4 4 4 5KlADH I 2.8 5.8=10 2.1=10 n.i. – – 0.11 8.1=10 7.3=10 n.i. – –4 4 5 5KlADH II 2.2 7.0=10 3.2=10 n.i. – – 0.18 1.2=10 6.6=10 n.i. – –

Mitochondrial4 5 3 3 5 7 4 5KlADH III 0.31 6.1=10 1.9=10 4.1 6.6=10 1.6=10 0.02 6.0=10 3.0=10 0.12 2.4=10 2.0=103 3 4 3 4 5 4 4KlADH IV 0.20 1.1=10 5.5=10 4.6 3.3=10 7.1=10 0.03 2.3=10 7.6=10 0.95 2.1=10 2.2=10

Ž . Ž y1. Ž y1 y1.n.i.: no interaction; K mM ; k min ; k rK min mM .m cat cat m


Table 3Alcohol preference of cytosolic and mitochondrial ADHs from

Ž .K. lactis. Relative reaction rates 258C of the KlADH isozymesfor the alcoholic substrates determined in 20 mM pyrophosphatebuffer, pH 8.5. The final concentrations of the alcohols were 100mM except for 2-mercaptoethanol which varied up to 300 mM.The values are expressed as percent of the rate obtained withethanol as substrate. Each value is the average of three determina-tions

Ž .Alcohol Relative reaction rates %

Cytosolic Mitochondrial

KlADH I KlADH II KlADH III KlADH IV

Ethanol 100 100 100 100Propanol 70 53 120 110Butanol 52 40 150 114Pentanol 26 28 140 57Hexanol 20 14 110 35iso-Propanol 0 0 32 152-Mercaptoethanol 0 0 0 0

chondrial isozymes were able to accept either NADq

or NADPq as coenzyme with a similar affinity, whileŽ .KlADH III exhibited a preference for NAD H with a

120r150-fold decrease of the k rK andcat mŽNADP.Ž .k rK , respectively Table 2 .cat mŽNADPH.

3.2. Influence of the buffer on the alcohol preferenceof the KlADH isozymes

The preference of the four KlADH isozymes fordifferent alcohols was investigated in the presence ofvarious buffers.

For each isozyme, the results are reported as rela-tive rates with respect to ethanol at the substrateconcentration of 100 mM. This value, in the case of

Žethanol, was in the range of concentrations 1–300. ŽmM where no inhibitory effect was observed see

.Section 2 . Table 3 reports the results obtained in 20Ž .mM pyrophosphate pH 8.5 where major differences

among the four isozymes were observed. However,although at a lower extent, the activation of bothmitochondrial isozymes was also observed with other

Ž .buffers, such as 50 mM phosphate pH 7.3 or 50Ž .mM Tris-HCl, pH 8.0. In pyrophosphate Table 3

the alcohol recognition properties of the cytosolicŽ .isozymes KlADH I and KlADH II were quite simi-

w xlar to those previously reported 10 . These activities

exhibited more restricted alcohol substrate prefer-Žences than the mitochondrial ones KlADH III and

.KlADH IV . In fact, KlADH III was able to oxidiselonger aliphatic primary alcohols, i.e. propanol, bu-tanol and pentanol and, to a lower extent, also hex-anol. KlADH IV showed a substrate preference pro-file similar to that of KlADH III, although lesspronounced. Interestingly, the mitochondrial isozymesacted also, even if with lower affinity, on the sec-ondary alcohol iso-propanol which instead was notrecognized as a substrate by the two cytosolicisozymes.

3.3. Aldehyde substrate specificity of the KlADHisozymes

The behaviour of KlADH isozymes towards vari-Žous aldehydic substrates was rather similar data not

.shown . In fact, as far as the reaction rate is con-cerned, acetaldehyde was the best substrate for thefour KlADH isozymes. Reaction rates decreased veryquickly with the elongation of the substrate aliphaticchain, especially in the case of the mitochondrialforms. In particular, benzaldehyde and glyceralde-hyde were not substrates for any isozyme, whileformaldehyde and salicylaldehyde were very poorsubstrates only for KlADH II and KlADH III.

3.4. Effect of 2-mercaptoethanol on the actiÕity of theKlADH isozymes

2-Mercaptoethanol did not appear to be a substratefor the cytosolic or the mitochondrial KlADH

Ž .isozymes Table 3 . However, a peculiar behaviourwas observed when KlADHs were pre-incubated in

Ž .Tris-HCl pH 8.0 with 2-mercaptoethanol for 5 minŽ .at 258C Fig. 1 . In fact, the activities of either

cytosolic or mitochondrial KlADH IV isozymes were3- and 2-fold enhanced by increasing up to 50 mMthe thiol concentration, whereas the activity ofKlADH III isozyme was unaffected by the presence

Ž .of 2-mercaptoethanol Fig. 1 . These results sug-gested that 2-mercaptoethanol could reduce disulfide

Ž .bridge s in KlADH IV and cytosolic isozymes, whoseformation was responsible for the decreased activity.A similar pattern was also observed by using 20 mM

Ž . Ž .pyrophosphate pH 8.5 or 50 mM Hepes pH 7.3 asbuffers in the incubation mixtures.


Fig. 1. Effect of 2-mercaptoethanol on the activity of the KlADHŽ .isozymes. Aliquots of the cytosolic, KlADH I v , KlADH II

Ž . Ž . Ž .B , and mitochondrial, KlADH III ^ , KlADH IV I

isozymes were pre-incubated for 5 min at 258C with increasingŽ .concentrations 1–50 mM of 2-mercaptoethanol in 20 mM

TrisrHCl, pH 8.0. ADH activity was assayed in the oxidativedirection in the presence of NADq as a cofactor. The results areexpressed as percent of the rate obtained by pre-incubating eachisozyme in the absence of 2-mercaptoethanol.

3.5. N-terminal sequences

The N-terminus of both KlADH I and KlADH IIisozymes was found to be blocked. On the contrary,the N-terminus of the KlADH III and KlADH IVisozymes was found to be Ala and Asn, respectively,revealing that the proteolytic cleavage of the protein

Ž .precursors occurred at a Leu residue. In Fig. 2 boldthe N-terminal sequences of the mitochondrialisozymes, as determined by automated Edman degra-dation of the purified proteins, are reported. Thecleavage site of the precursor of KlADH IV had been

w xcorrectly predicted by Saliola et al. 4 , by applyingthe empirical rule based on the observations of Hartl

w xet al. 29 . According to the latter authors, the prote-olytic processing of the precursor of mitochondrial

Fig. 2. Comparison of the yeast mitochondrial ADH precursorŽ .sequences. The sequences from K. lactis KlADH and S. cere-

Ž .Õisiae SADH were aligned according to the N-terminus of theŽ .mature proteins in bold . Arg residues preceding the Leu after

which the proteolytic cleavage occurs are underlined.

proteins requires the presence of a basic residue, Lysor Arg, in position y2, the occurrence of a Ser inposition q2 and Ser or Thr in position q3 withrespect to the cleavage site. Besides KlADH IV,these requirements have been also found in the pre-cursor of the mitochondrial alcohol dehydrogenaseŽ . w xSADH III from S. cereÕisiae 30 . The cleavage siteoccurring at the KlADH III precursor does not strictlyconform to the above reported rule, mostly because

Ž .of the Arg in position y3 Fig. 2 .

3.6. Molecular mass and subunit composition of theKlADH isozymes

The native KlADH isozymes, as indicated bysize-exclusion chromatography, exhibited an averagemolecular mass of 150 kDa, with elution volumessimilar to that observed for the commercial yeastalcohol dehydrogenase. Under denaturing conditionsŽ .SDS-PAGE the molecular mass of the four KlADH

Žisozymes ranged between 37–38 kDa data not.shown , well in agreement with the values calculated

from the gene deduced sequences, thus suggestingthat each isozyme consisted of four apparently identi-cal subunits.

4. Discussion

The four ADH isozymes from K. lactis have beenpurified to homogeneity and characterized. They areorganized as homotetramers with an average molecu-lar mass of 150 kDa. Both the cytosolic KlADHsresulted to be N-terminally blocked; in this respect,they were similar to other cytosolic ADHs whichfrequently have their N-terminus acetylated. Con-versely, the N-terminal sequence of the mitochondrialisozymes, KlADH III and KlADH IV, allowed theidentification of the site of cleavage of mitochondrialprecursors after a Leu residue, probably by the actionof the same proteinase. The calculated molecularmass of the KlADH III and IV monomers was 36 884

Ž . Ž .Da 349 residues and 37 365 Da 350 residues ,respectively.

The cytosolic KlADHs showed an overall substratew xspecificity close to the S. cereÕisiae ADH I 24 , with

a preference for ethanol, whereas the enzyme activitydecreased quickly with the elongation of the aliphatic


chain of the primary alcohol substrates. In contrast,the mitochondrial KlADH isozymes, in particularKlADH III, increased their activity with the chainelongation of the primary alcohols, reaching the max-imum relative rate with butanol as substrate. Thiseffect, which was mostly observed in pyrophosphateand, to a lower extent, in other buffers, is suggestiveof an interaction of pyrophosphate with some ex-posed regions or with areas surrounding the active-sitepocket of the mitochondrial proteins. As a conse-quence, the catalytic cavity of the mitochondrialKlADHs would result more accessible to differentsubstrates than that of the cytosolic ones. This hy-pothesis is also supported by the results of Green et

w xal. 21 who demonstrated, by using site-directedmutagenesis experiments, that the substrate speci-ficity of S. cereÕisiae ADH I on long chain primaryalcohols could be broadened by enlarging its sub-strate pocket.

The K values of the cytosolic KlADHsm Žethanol.were greater than those observed for the mitochon-drial isozymes and comparable to the K valuem

reported for the cytosolic ADH from S. cereÕisiaew x24 . Conversely the k rK values of thecat m Žethanol.cytosolic KlADHs were lower than that of KlADHIII and similar to that of KlADH IV, leading to theconsideration that KlADH III exhibited the highestaffinity for ethanol. Our data confirm the in vivo

w xobservations previously reported by Saliola et al. 3that KlADH I, similarly to SADH I, would representthe fermentative enzyme which is preferentially ex-pressed in glucose-grown cells and seems mainlyinvolved in the ethanol production. It is also reason-able to suppose that KLADH II plays a role inethanol production, since its affinity for both ac-etaldehyde and ethanol is comparable to that ob-served in the case of KlADH I. In addition, whileKlADH III displayed a marked preference for ac-etaldehyde, KlADH IV exhibited almost the sameaffinity for ethanol or acetaldehyde, thus indicatingthat it can accept indifferently one or the other ofthese two substrates, probably in response to varia-tions of intra-mitochondrial conditions.

One of the more interesting features of the mito-chondrial KlADH isozymes was that they can accept,at variance with the cytosolic KlADHs and most of

Ž .qthe ADHs from other sources, either NAD P orŽ .NAD P H as coenzymes with a slight preference of

KlADH III for the reduced forms. Moreover, whilemitochondrial KlADH IV bound almost equally wellNADq or NADPq, the cytosolic isozymes exhibited

Ž .a strict specificity for NAD H . This diversity shoulddepend on different arrangements of the active-siteregions of the two isozyme pairs.

w xRecently, Gonzales Siso et al. 31 have hypothe-sised the existence in K. lactis of a dehydrogenase,probably localized on the external membrane of mito-chondria, which can fulfil the role of reoxidizing thecytosolic NADPH produced in the pentose phosphatepathway more abundantly in this yeast as comparedto S. cereÕisiae. At present, the identification of thisactivity with the mitochondrial KlADH isozymes canbe considered only as a speculation, since their pre-cise localization within mitochondria has not yet beendetermined. In any case, the peculiar affinity ofKlADH III and KlADH IV for NADPH could satisfythe requirement for reoxidation of this coenzyme inK. lactis.

From a structural standpoint, the naturally occur-ring capability of mitochondrial KlADHs of binding,and in some case almost equally well, both NADq

and NADPq resulted to be rather surprising. In fact,Asp-223 of horse liver ADH, which is a residueconserved in all the NAD-dependent ADHs, is thoughtto be responsible for the selection of this coenzymew x16–18 . Its replacement by glycine, converted both

w xthe ADH of T. brockii 32 and ADH I of S. cere-w xÕisiae 22 in a NADP-dependent enzyme, therefore

highlighting the critical role of Asp-223 in determin-ing coenzyme specificity. By comparing the ADHamino-acid sequences from K. lactis, S. cereÕisiae

Ž .and horse liver Fig. 3 , we observed that in mito-chondrial and cytosolic KlADHs the residue corre-sponding to Asp-223 of horse liver ADH is strictlyconserved, thus apparently contradicting the previous

w xconclusions 16,18 . On the other hand, it should benoticed that also in the case of glyceraldehyde-3-phosphate dehydrogenase the substitution of the Aspresidue, which was supposed to be responsible for theNAD selective affinity, did not improve significantly

Ž . w xthe affinity for NADP H 33 . Our observations sug-gest that Asp-223 of ADH should not be consideredmore critical than the other amino-acid residues lin-ing the coenzyme binding pocket, at least for whatconcerns the mitochondrial KlADH isozymes. On theother hand, on the basis of the structural model of the


w xcoenzyme binding site reported by Fan et al. 22 , weobserved that other residues such as His-47, Ala-201,Gly-225 and in particular Lys-228, which togetherwith Asp-223 forms hydrogen bonds with 2X- or3X-hydroxyl groups of the adenosine ribose of coen-zyme, are strictly conserved in the sequences of both

Ž .SADH I, and KlADHs Fig. 3 . Thus, a possibleexplanation is that the coenzyme binding sites of themitochondrial KlADHs, perhaps owing to a major

Ž .flexibility, are more accessible to NADP H than

those of the cytosolic isozymes. This parallels whathas been observed for the alcohol binding sites.

Whereas 2-mercaptoethanol was able to selectivelyinhibit the alcohol dehydrogenase from horse liver

w xbut not the commercial yeast ADH 19 , the activitiesof both cytosolic and KlADH IV isozymes were 3-

Ž .and 2-fold enhanced by this thiol compound Fig. 1 .On the basis of the similarities observed among theADH amino-acid sequences from K. lactis, S. cere-Õisiae and horse liver, the cysteine residues binding

Ž . Ž . Ž . Ž .Fig. 3. Comparison of cytosolic c and mitochondrial m ADH amino-acid sequences from K. lactis KlADH , S. cereÕisiae SADHŽ . w xand horse liver HLADH . The sequences were aligned according to Jornvall et al. 17 . Numbering refers to the horse liver sequence. The¨

Ž . Ž .cysteine residues binding the catalytic empty circles and the structural filled circles zinc ions in horse liver ADH are boxed, as well asŽ . Ž .the additional cysteines present in KlADHs filled squares and Asp-223 asterisk .


Ž .either the catalytic Fig. 3, empty circles , or theŽstructural zinc ions of horse liver ADH Fig. 3, filled

. w xcircles resulted to be highly conserved 17,18 .Moreover, both cytosolic and KlADH IV isozymeshad an additional cysteine, i.e. Cys-301 or Cys-236,

Ž .respectively Fig. 3, filled squares . Therefore, aninvolvement of the extra-Cys residue in a disulfidebridge between the subunits of each tetramer canprobably occur.

Since the NAD-dependent alcohol dehydrogenasesso far studied have been grouped into various classes,the mitochondrial KlADH isozymes, owing to theirpeculiar coenzyme specificity, could fall into a newclass. The reported results demonstrate that mito-chondrial and cytosolic KlADHs had a common evo-lutionary origin and then diverged until they acquiredthe properties above described, thus confirming pre-vious suggestions on the evolution of the yeast ADH

w xgenes 10,17,34 .Finally, the different capability of the KlADH

isozymes of accepting both NADq and NADPq as acofactor provides also a useful method for discrimi-nating mitochondrial from cytosolic KlADHs.

Acknowledgements

We thank Dr. N. Franceschini for HPLC gel-filtra-tion analyses. This work was supported in part bygrants from the Italian Ministero dell’Universita e`della Ricerca Scientifica e Tecnologica.

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Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis

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