differences in the roles of conserved glutamic acid residues in the

Differences in the roles of conserved glutamic acidresidues in the active site of human class 3and class 2 aldehyde dehydrogenases

CRAIG J. MANN and HENRY WEINERDepartment of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153

~Received February 19, 1999;Accepted June 4, 1999!

Abstract

Although the three-dimensional structure of the dimeric class 3 rat aldehyde dehydrogenase has recently been published~Liu ZJ et al., 1997,Nature Struct Biol 4:317–326!, few mechanistic studies have been conducted on this isoenzyme.We have characterized the enzymatic properties of recombinant class 3 human stomach aldehyde dehydrogenase, whichis very similar in amino acid sequence to the class 3 rat aldehyde dehydrogenase. We have determined that therate-limiting step for the human class 3 isozyme is hydride transfer rather than deacylation as observed for the humanliver class 2 mitochondrial enzyme. No enhancement of NADH fluorescence was observed upon binding to the class 3enzyme, while fluorescence enhancement of NADH has been previously observed upon binding to the class 2 isoen-zyme. It was also observed that binding of the NAD cofactor inhibited the esterase activity of the class 3 enzyme whileactivating the esterase activity of the class 2 enzyme. Site-directed mutagenesis of two conserved glutamic acid residues~209 and 333! to glutamine residues indicated that, unlike in the class 2 enzyme, Glu333 served as the general base inthe catalytic reaction and E209Q had only marginal effects on enzyme activity, thus confirming the proposed mechanism~Hempel J et al., 1999,Adv Exp Med Biol 436:53–59!. Together, these data suggest that even though the subunitstructures and active site residues of the isozymes are similar, the enzymes have very distinct properties besides theiroligomeric state~dimer vs. tetramer! and substrate specificity.

Keywords: aldehyde dehydrogenase; catalytic residues; NAD~H! conformation; rate-limiting step; site-directedmutagenesis

Isoenzymes of aldehyde dehydrogenases~ALDH ! play importantroles in intermediary metabolism from organisms as diverse asbacteria and humans. In humans, the class 2 mitochondrial isoen-zyme ~hALDH2! is involved in the oxidation of acetaldehyde toacetate during the metabolism of ethanol~Svanas & Weiner, 1985!.The class 1 cytosolic isozyme~hALDH1! is thought to be involvedin the metabolic conversion of retinaldehyde to retinoic acid~Kit-son & Blythe, 1999!, while the physiological substrates for theclass 3 ~hALDH3! isoenzymes are unknown. However, bothhALDH1 and hALDH3 are thought to be involved in the metabolicdetoxification of the important chemotherapeutic and immunosup-pressive drug, cyclophosphamide~Dockham et al., 1992; Sreerama& Sladek, 1993!.

The crystal structure of dimeric rat liver ALDH3~Liu et al.,1997! has provided information to complement structural data avail-

able on the tetrameric enzymes of bovine ALDH2~Steinmetzet al., 1997!, sheep liver ALDH1~Moore et al., 1998!, and codliver betaine ALDH ~Johansson et al., 1998!. In each of theseenzymes, there are three large domains; a catalytic domain, a co-enzyme binding domain, and an oligomerization domain. The sub-strate specificity of each of the different isozyme forms appears todetermined by amino acids lining the tunnel pointing toward theactive site~Moore et al., 1998!. These residues might help todiscriminate between aromatic, aliphatic, and long chain hydro-carbon aldehydes.

Previous work from this laboratory has suggested that the rate-limiting step for horse liver ALDH2 was deacylation~Fig. 1! ~Feld-man & Weiner, 1972; Weiner et al., 1976!. This was later observedfor human ALDH2 as well~Wang & Weiner, 1995!. Chemicalmodification ~Pietruszko et al., 1991! and site-directed mutagen-esis experiments~Farrés et al., 1995! have demonstrated that Cys302is the active site cysteine. Glu268 has been identified as the generalbase that interacts with the active site cysteine or water duringenzyme catalysis~Wang & Weiner, 1995!. More recently, some ofthe other conserved residues have been mutated~Li et al., 1997;Sheikh et al., 1997!. From these studies it was found that mutationof Glu399, a residue that interacts with the ribose moiety of NAD,to glutamine converted the rate limiting step from deacylation tohydride transfer~Li et al., 1997!.

Reprint requests to: Dr. Henry Weiner, Department of Biochemistry,Purdue University, West Lafayette, Indiana 47907-1153; e-mail: [email protected].

Abbreviations:ALDH, aldehyde dehydrogenase; EDTA, ethylenediamine-tetraacetic acid; hALDH1, human class 1 aldehyde dehydrogenase; hALDH2,human class 2 aldehyde dehydrogenase; hALDH3, human class 3 alde-hyde dehydrogenase; NAD, nicotinamide adenine dinucleotide; NPA,p-nitrophenyl acetate; PCR, polymer chain reaction; SDS, sodium dodecylsulfate.

Protein Science~1999!, 8:1922–1929. Cambridge University Press. Printed in the USA.Copyright © 1999 The Protein Society

1922

A recent proposal for the catalytic mechanism of ALDH3~Hempelet al., 1999! suggested that the conserved Glu333, correspondingto Glu399 in the class 2 isoenzyme, is the essential general base,rather than being involved in coenzyme binding. Comparison ofthe crystal structure data of rat liver ALDH3~Liu et al., 1997! tothat of bovine ALDH2~Steinmetz et al., 1997! suggests globalstructural similarities between the enzymes but also indicates dif-ferences in the conformation of bound NAD. This study describesour efforts to characterize some of the similarities and differencesbetween the isozyme forms to gain a better understanding of therole of conserved residues in hALDH3.

Results

Expression and purification of hALDH3 from bacterial cells

Hsu et al.~1992! first described the cloning and expression ofhuman stomach ALDH3 inEscherichia coli. These investigatorsreported that only 10–15% of the recombinantly expressed proteinremained soluble. We have observed similar levels of solubly ex-pressed wild-type and mutant proteins described in this study~datanot shown!. In addition, while they did not purify the recombinantenzyme to homogeneity, their initial kinetic experiments suggestedthat the substrate specificity of this enzyme favored benzaldehydeover hexanal or propionaldehyde~Hsu et al., 1992!. This prefer-ence for benzaldehyde has been also described for ALDH3 puri-fied from human liver~Yin et al., 1989! and human stomach~Wanget al., 1990; Sreerama & Sladek, 1993!. It is this substrate prefer-ence of the enzymes in the ALDH superfamily that has been usedto help distinguish between each of the different isozyme forms.

The procedure described for the purification of hALDH3 fromhuman stomach~Sreerama & Sladek, 1993! was simplified in thisstudy by introducing a His-tag sequence at the C-terminus of theprotein. Lysed cell extracts were then passed over a Chelating-Sepharose column to capture the recombinant hALDH3 proteinsfollowed by the final purification step using Blue-Sepharose. Wild-type enzyme hALDH3 purified using this procedure resulted inpreparations having similar specific activities to those described bySreerama and Sladek~1993! for hALDH3 purified from stomach

tissue. Purification of the E333Q mutant required a different ap-proach because it was found that elution of the protein from theChelating–Sepharose column with imidazole and subsequent dial-ysis to remove the imidazole and NaCl resulted in the precipitationof the partially purified protein. To prevent this problem, elution ofthis mutant enzyme from the column employed a 0–20 mM EDTAgradient followed by dialysis in high salt to remove only the EDTA.Only marginal characterization was conducted on the partiallypurified E333Q mutant enzyme because of its lack of enzymeactivity and solubility problems at low ionic strength.

Wild-type enzyme characterization

Initial kinetic characterization of the purified wild-type hALDH3showed that substrate inhibition occurred at concentrations above400 mM benzaldehyde~data not shown!. For experiments de-scribed in this study, conditions were chosen such that sufficientdata were collected below this upper limit in benzaldehyde con-centration. As such, the reported kinetic parameters have to bedesignated as apparentKm ~Km,app! andVmax ~Vmax,app! values.

Rate limiting step

To determine the rate limiting step for hALDH3, we incubatedpurified wild-type enzyme with several benzaldehyde analogs toobtain reaction velocities~Table 1!. For the most electron donatingsubstrate, p-methoxybenzaldehyde, the reaction velocity increasedalmost threefold over the benzaldehyde velocity, while for themost electron-withdrawing substrate, p-nitrobenzaldehyde, the re-action velocity decreased 10-fold from that of benzaldehyde. Thesedata suggested that the rate limiting step for this enzyme washydride transfer. To further confirm this conclusion, wild-type en-zyme was also incubated with benzaldehyde or deuterated benz-aldehyde. As shown in Table 2, the velocity obtained with deuteratedbenzaldehyde was approximately twofold lower than that obtainedwith benzaldehyde, clearly indicating a primary isotope effect.This is in contrast to hALDH2 where no primary isotope effect wasfound ~Li et al., 1997!. Taken together, these data suggest that therate limiting step for hALDH3 is hydride transfer rather than de-acylation as seen for hALDH2~Wang & Weiner, 1995!.

A

B

Fig. 1. Reaction mechanisms for ALDH enzyme activities.A: Reaction scheme for the dehydrogenase activity. E-SH indicates theenzyme with the active site cysteine~Cys243 in ALDH3 and Cys302 in ALDH2! as the nucleophile. Aldehyde substrate binds to theE-NAD complex to form the thiohemiacetal intermediate, which is then oxidized to an acyl intermediate. Deacylation then takes place.B: Reaction scheme for the esterase activity. The nitrophenyl acetate~NPA! binds to the enzyme and nitrophenol~NP! is releasedduring the deacylation step

Nonfunctional conservation of active site residues in human ALDH3 1923

NADH binding studies

To characterize and compare the cofactor binding of hALDH3 andhALDH2, we examined the fluorescence changes of NADH uponbinding to each enzyme. This type of experiment has previouslyshown an enhancement in the fluorescence of NADH when boundto hALDH2 ~Wang & Weiner, 1995! and other dehydrogenasessuch as horse liver alcohol dehydrogenase~Iweibo & Weiner, 1975!and bovine liver glutamate dehydrogenase~Delabar et al., 1982!.In the case of hALDH3, no NADH fluorescence enhancement wasobserved when cofactor bound to the enzyme~data not shown!,suggesting that the hydrophobic environment around the boundcofactor does not significantly effect the fluorescence of NADH.This lack of NADH fluorescence enhancement upon binding hasalso been observed for other dehydrogenases such as glyceraldehyde-3-phosphate dehydrogenase~Scheek et al., 1979!.

Esterase activity and cofactor inhibition

In addition to the dehydrogenase activity of ALDH, this enzymealso exhibits an inherent esterase activity~Feldman & Weiner,1972! that was also used to characterize the wild-type and mutantenzymes. Interestingly, the esterase activity of hALDH2 was ac-tivated by low levels of NAD~Sheikh et al., 1997!. In horse liverALDH2, this effect was attributed to NAD increasing the nucleo-philicity of Cys302 ~Takahashi & Weiner, 1981!. These experi-ments were repeated with hALDH3 for comparison to hALDH2~Fig. 2!. As was seen previously, the esterase activity of hALDH2was enhanced approximately 2.5 times with the addition of up to

200mM NAD. In contrast, the same concentration of NAD nearlyabolished the esterase activity of hALDH3 with aKis 5 120 mM.These data, along with the lack of NADH fluorescence enhance-ment, suggest that the conformation of NAD, when bound to theenzyme, is clearly very different between the hALDH3 and hALDH2isozyme forms and is consistent with the NAD conformationaldifferences observed in the crystal structures for the class 3~Liuet al., 1997! and class 2~Steinmetz et al., 1997! enzymes.

Role of conserved glutamic acid residuesin the dehydrogenase activity

While the crystallographic data clearly indicate a difference in thebound conformation of NAD in rat ALDH3~Liu et al., 1997! andbovine ALDH2 ~Steinmetz et al., 1997! isozymes, several con-served residues in their respective active sites have similar loca-tions ~Fig. 3!. These residues include Glu333, Asn114, Cys243,and Glu209 for ALDH3 and the corresponding residues in ALDH2~Glu399, Asn169, Cys30, Glu268!. We have used site-directedmutagenesis to ascertain the role of the conserved active site glu-tamic acid residues in hALDH3 by singly replacing Glu209 andGlu333 with Gln.

Quantitative activity data were obtained for the purified prepa-rations of the wild-type hALDH3 and the E209Q mutant enzyme.As shown in Table 3, for the wild-type enzyme theKm,app forbenzaldehyde and NAD were 220 and 7.8mM, respectively. Forthe E209Q mutant enzyme, theKm,app value for benzaldehyde wassimilar to wild-type ~580 mM !. In contrast, theKm,app for NADwas 10-fold larger~61 mM ! and thekcat value was also slightlydecreased compared to wild-type enzyme suggesting that this mu-tation affected the ability of this enzyme to interact with NAD.

The dehydrogenase activity of the partially purified E333Qmutant enzyme was assessed qualitatively by first estimating theamount of enzyme in the preparation using Coomassie-stainedSDS gels. Then activity assays were conducted underVmax,app

conditions~1 mM NAD and 400mM benzaldehyde!. A relativespecific activity of,1% compared to wild-type enzyme was ob-

Table 1. Benzaldehyde analogs as substrates for hALDH3

X-benzaldehyde

sa X 5 Vb

20.268 p-CH3O- 2.720.170 p-CH3- 0.9

0.0 H- 1.010.551 p-CF3- 0.110.778 p-NO2- 0.1

as ~the substituent constant! is defined as a measure of the ability of thesubstituent to change the electron density at the reaction center~Hine,1956!. Groups with negative substituent constants are electron-donating,while those with positive substituent constants are electron-withdrawing.

bStandard assay conditions~Farrés et al., 1994! were used to obtainmaximum velocity data that were then normalized to the velocity obtainedfor benzaldehyde under the same assay conditions.

Table 2. Primary 2H isotope effects on ALDH activityusing benzaldehyde as a substrate

V~mmol0min0mg! VH VD VH0VD

hALDH3 17 8.2 2.1hALDH2a 0.17 0.17 1.0

aData taken from Li et al.~1997!.

Fig. 2. Effect of NAD addition on the esterase activity of aldehyde de-hydrogenase. Data for wild-type hALDH3~l!, E209Q mutant~▫!, andwild-type hALDH2 ~m! are shown. Samples were diluted with 100 mMsodium phosphate pH 7.4 and assayed in the presence of 25mM p-nitrophenylacetate as a substrate.

1924 C.J. Mann and H. Weiner

tained~Table 3!, suggesting that a dramatic loss of enzyme activityin the E333Q mutant has taken place. There was so little activityin the mutant preparation that it was not practical to attempt todetermine the kinetic constants.

Role of conserved glutamic acid residuesin the esterase activity

As shown in Table 4, the p-nitrophenyl acetateKm value for theE209Q mutant is approximately 30-fold higher than that observedfor wild-type hALDH3. The esterase activity of the E209Q mutantwas only partially inhibited by NAD~;50%! compared to nearlycomplete inhibition of the wild-type enzyme~Fig. 2!. The com-bined dehydrogenase and esterase activity data strongly suggestthat Glu209 in hALDH3 cannot be considered the general base, butrather that it appears that Glu333 could be performing this essen-tial function in this enzyme.

Discussion

The recently reported structure of rat ALDH3~Liu et al., 1997! hasgreatly facilitated the comparison of this dimeric enzyme to that ofthe crystallized bovine ALDH2~Steinmetz et al., 1997! and thesubsequent analogous comparisons of the hALDH3 and hALDH2.As shown in Figure 3, direct comparison of the active sites of thetwo crystallized enzymes indicates that similar residues are presenteven though these two enzymes prefer different aldehyde sub-strates. This difference is probably due to accessibility of the activesite as governed by residues in the aldehyde substrate channel~Moore et al., 1998! rather than changes in specific active siteresidues. It is interesting to note that while the active site Cysresidues~presumed to be 243 in class 3 and shown to be 302 inclass 2~Farrés et al., 1995!, respectively! are positioned spatiallysimilar to conserved glutamic acid residues~2090268! and ~3330399!, the NAD molecules appear to be in different orientationswithin each isozyme. Examination of the importance of NAD bind-ing will be discussed in detail below. Based on site-directed mu-tagenesis experiments~Wang & Weiner, 1995!, the role of thegeneral base in hALDH2 has been assigned to Glu268. In contrast,information based on the crystallographic data suggested that Glu333might serve as the general base in ALDH3~Liu et al., 1997!. Aproposed mechanism for ALDH3 has also suggested that Glu333serves as the catalytic base in this enzyme~Hempel et al., 1999!.In other experiments, mutation of the corresponding residue inALDH2 ~Glu399! has been shown to change the rate limiting stepfor the dehydrogenase reaction from deacylation to hydride trans-fer without dramatically altering the specific activity of either en-zyme reaction~Li et al., 1997!. With this as a starting point, webegan our characterization of human stomach ALDH3.

It is apparent from the data that while the rate-limiting step forhALDH2 is deacylation~Wang & Weiner, 1995!, the rate-limitingstep for hALDH3 is hydride transfer~Tables 1, 2!. This switch inthe rate-limiting step may be related to the conformational differ-ence of bound NAD and to the relative flexibility of the nicotin-amide ring. Excessive movement of the ring would keep it out ofthe proper position for hydride transfer and thus make this step inthe enzyme reaction rate limiting. Whether this cofactor flexibilityhas come about because of changes in the local environment withinthe enzyme, changes in substrate preferences or their availability,or some other factor related to evolutionary pressure has yet to bedetermined.

Since it appeared that the rate-limiting step was different forhALDH3 and hALDH2, it was also of interest to examine thepotential conflicting proposed roles of the active site glutamic acid

Fig. 3. Comparison of active site residues in ALDH3 and ALDH2. Theactive site of dimeric rat class 3 ALDH~Liu et al., 1997! ~stick model! andthe active site of tetrameric bovine class 2 ALDH~Steinmetz et al., 1997!~wireframe model! were overlaid and aligned based on the orientation ofthe cysteine residues~234, 302, in yellow! and glutamic acid residues~209and 268, in cyan! using Rasmol~Sayle & Milner-White, 1995!. The NADcofactor~in red! is also shown to highlight orientation differences betweenthe isozyme forms. Additional active site residues include Glu333 andGlu399 ~in blue! and Asn114 and Asn169~in green!.

Table 3. Kinetic parameters of the dehydrogenase activityof hALDH3 and mutant enzymes

Km,app ~mM !

Benzaldehyde NADkcat

~min21! % SAa

hALDH3 220 7.8 54 100E209Q 580 61 47 87E333Q N.D.b N.D. N.D. ,1

aPercent specific activity was calculated for activity assays underVmax,app

conditions~1 mM NAD and 400mM benzaldehyde!.bN.D. 5 not determined.

Table 4. Kinetic parameters of the esterase activityof hALDH3 and mutant enzymesa

NPA Km

~mM!kcat

~min21!

hALDH3 0.2 86E209Q 6.8 240E333Q N.D.b N.D.

aAssays were performed in 100 mM sodium phosphate~pH 7.4! usingNPA as a substrate.

bN.D. 5 not determined.


residues. Site-directed mutagenesis experiments were used to sin-gly convert the conserved glutamic residues~209 and 333, corre-sponding to Glu268 and Glu399 in hALDH2! to glutamine residues.While it appeared that the activity of the E209Q mutant enzymewas only slightly different from wild-type enzyme, the E333Qenzyme had little to no residual enzyme activity. However, thelatter also appeared to be considerably less stable than wild-typeenzyme. This nearly complete loss of both enzyme activities hadbeen previously used to implicate Glu268 in hALDH2 as the gen-eral base for this enzyme~Wang & Weiner, 1995!. This suggeststhat the role of these two glumatic acid residues~Glu2090Glu268!are different in these two enzymes. The current ALDH3 structure~Liu et al., 1997! precludes the assignment of a specific role ofGlu209, such as ribose binding, as shown for Glu399 in the ALDH2structure~Steinmetz et al., 1997!. Finding that the E333Q mutantwas essentially inactive is consistent with its role being that of ageneral base. The lack of sufficient solubility of this mutant hin-dered its further characterization.

Examination of the esterase activity of these enzymes also showeddifferences between the two isozyme forms. The overall esteraseactivity of hALDH3 seems to be similar to hALDH2 even thoughthe Km for NPA is 45-fold lower for hALDH3. This may be re-flective of the substrate specificity of hALDH3 and suggests thataromatic esters, as well as aromatic aldehydes are better substratesfor this enzyme. The binding of NAD to hALDH3 actually inhibitsesterase activity~Kis5120mM !, whereas NAD stimulates hALDH2esterase activity. The rate-limiting step for the esterase reactioncatalyzed by the class 3 enzyme is not known but is either acyla-tion, as with the class 2 enzyme, or deacylation~Fig. 1!. Theactivation found with the class 2 enzyme had been postulated to bea result of an increased nucleophilicity of Cys302~Takahashi &Weiner, 1981! caused by the binding of coenzyme. It is not pos-sible to speculate how the presence of NAD inhibits the esterasereaction of hALDH3 or why the E209Q mutation results in adiminution of this inhibition. Independent of the mechanism, theobservation is consistent with different affects of NAD binding tothe active site regions of the two isoenzymes.

Further characterization of wild-type hALDH3 as compared tohALDH2 showed that while binding of NADH to hALDH2 gavean enhancement in NADH fluorescence~Wang & Weiner, 1995!,in the case of hALDH3, no fluorescence enhancement was ob-served~data not shown!. This was another indication that the con-formation of NADH when bound to hALDH3 was different fromthat normally seen in hALDH2. Reports in the literature on thebinding of NADH and analogs of NAD to other dehydrogenases~Luisi et al., 1975; Hones et al., 1986! suggest that changes in theirfluorescence properties can be attributed to conformational move-ment of the nicotinamide ring rather than the adenosine moiety.Comparison of the bound NAD or NADH structures in severaldehydrogenase crystal structures indicates that the accessibility ofthe nicotinamide ring is quite variable~Fig. 4! and supports theidea that the fluorescence properties of these enzymes might bedifferent. In addition, movement of this portion of the cofactormight also depend on interaction with solvent~i.e., ALDH3 andglyceraldehyde 3-phosphate dehydrogenase! and the fluidity of theprotein structure surrounding the nicotinamide ring. Those de-hydrogenases with constrained or buried nicotinamide rings, andhence more fluorescent, would have a tendency to be less mobileand would have to rely on initial conformational changes of theprotein structure to allow for subsequent ring movement~i.e.,ALDH2 and lactate dehydrogenase!.

Substrate specificity of the class 3 isozyme makes it appear thatthis enzyme form prefers large aromatic substrates while for class2 simple aliphatic aldehydes, such as acetaldehyde and propional-dehyde, are good substrates. The structures of the binary com-plexes between enzyme and NAD show that the pocket surroundingthe nicotinamide ring is more open in the case of the class 3enzyme while the ring is more buried in the class 2 form. Theopenness, and hence accessibility to water, can be used to explainwhy the fluorescence of NADH is not enhanced when bound to theclass 3 enzyme just as it is in glyceraldehyde 3-phosphate de-hydrogenase. It can be predicted that in the ternary complex thepocket closes. With the class 2 enzyme, the pocket might be morehydrophobic so that there will be less conformational change in theternary complex. To date, there is no report of the structure of aternary complex of ALDH but it is known that for the class 2enzyme there is no conformational difference between the apo andbinary complex~T.D. Hurley, unpubl. data!.

Consistent with the assumption that movement of the proteinoccurs in the ternary complex is the multiple conformations of theNAD cofactor in the crystal structure of the tetrameric sheep cy-tosolic ALDH ~Moore et al., 1998!. The lack of a ternary complexstructure for any of the tetrameric enzymes precludes us fromdefining what the true conformation of NAD should be for opti-mum catalytic efficiency for these isozymes. For ALDH3, it ap-pears that alignment of the NAD cofactor in the active site~Liuet al., 1997! positions it within close proximity of the general baseGlu333, supporting our data. If, on the other hand, Glu209 wasactually the general base, the NAD conformation would have to bealtered to move closer to this residue. The fact that our E209Qmutant was only slightly less active than wild-type further sup-ports the improbability that this residue functions as the catalyticbase.

In summary, these data suggest that even though the subunitstructures and active site residues of ALDH3 and ALDH2 aresimilar, the enzymes have very distinct properties besides theiroligomeric state~dimer vs. tetramer!. For hALDH3, these traitsinclude hydride transfer as the rate limiting step, Glu333 acting asthe general base~confirming the proposed mechanism~Hempelet al., 1999!! and the inhibition of esterase activity by NAD. Forthese two enzymes, it appears that conservation of active site res-idues does not necessarily predict conserved functional roles. Untilthe ternary complexes are solved for these two enzymes or otheraldehyde dehydrogenases are characterized, the precise conforma-tion of the enzyme and the bound NAD will remain open fordiscussion. A more general mechanism for the enzymatic reactioncatalyzed by aldehyde dehydrogenase awaits the characterizationof additional members of this large family of enzymes.

Materials and methods

Materials

NAD, NADH, p-nitrophenyl acetate were purchased from Sigma~St. Louis, Missouri!; benzaldehyde and benzaldehyde analogs werepurchased from Sigma or Aldrich Chemical Co.~Milwaukee, Wis-consin!; a-@2H#benzaldehyde~C6H5CDO! was purchased fromCambridge Isotope Laboratories, Inc.~Woburn, Massachusetts!;SequiTherm Excel II DNA sequencing kit was purchased fromEpicentre Technologies~Madison, Wisconsin!; restriction endonu-cleases were purchased from New England Biolabs~Beverly, Mas-sachusetts! or Promega Corp.~Madison, Wisconsin!; QIASPIN


miniprep plasmid purification and QIAEX II gel extraction kitswere purchased from Qiagen~Chatsworth, California!; @a-35S#dATPwas purchased from Amersham Corp.~Piscataway, New Jersey!;synthetic oligonucleotides used for sequencing and mutagenesiswere purchased from Integrated DNA Technologies, Inc.~Coral-ville, Iowa!.

Cells and plasmids

A plasmid containing the cDNA clone of human stomach ALDH3~kindly provided by Dr. Alan Townsend, Wake Forest University!was used as the starting template for PCRs to introduce suitablerestriction sites and the amino acid sequence—H-W-H-H-H~Kasheret al., 1993! at the C-terminal end of the protein to aid in purifi-cation. The resulting PCR product was subcloned into the pT7-7

expression vector and transformed into theE. coli strain BL21~DE3! ~pLysS!. Protein expression was conducted as previouslydescribed~Zheng et al., 1993!.

Mutagenesis of hALDH3

Mutations were introduced into the hALDH3 gene using syntheticoligonucleotides and PCR techniques previously described~Hoet al., 1989!. Mutations were confirmed by double-stranded DNAsequencing using a thermocycler sequencing kit~Epicentre Tech-nologies! according to the manufacturer’s protocol.

Purification of native and mutant enzymes

Recombinantly expressed enzymes were initially purified on aChelating–Sepharose~Pharmacia, Uppsala, Sweden! column

Fig. 4. Comparison of nicotinamide ring exposure when bound to dehydrogenases. Residues within a 10 Å shell~shown as solidsurface! around each of the bound NAD cofactors~magenta! in the dehydrogenase structures are shown. Hydrophobic~gray!,hydrophilic~cyan!, basic~dark blue!, and acidic~red! residues are also indicated surrounding the NAD molecules.A: Rat ALDH3 ~Liuet al., 1997!. B: Bovine ALDH2 ~Steinmetz et al., 1997!. C: Bacillus stearothermophilus glyceraldehyde3-phosphate dehydrogenase~Skarzynski et al., 1987!. D: Dogfish lactate dehydrogenase~White et al., 1976!. This figure was generated using GRASP~Nichollset al., 1991!. Structures were superimposed and aligned using the adenine rings of each of the NAD molecules. All structures are shownusing this alignment with the exception that the lactate dehydrogenase structure has been rotated 458 about they-axis to better visualizethe NAD structure.


~1.6 3 10 cm! charged with zinc acetate as previously described~Kasher et al., 1993! and equilibrated with 50 mM sodium phos-phate pH 7.5, 500 mM NaCl. Wild-type hALDH3 or E209Q mu-tant enzymes were eluted from this column using a three columnvolume imidazole gradient~0–500 mM! in 50 mM sodium phos-phate pH 7.5, 500 mM NaCl. Fractions containing the wild-type orE209Q enzymes were identified by activity assays and SDS gels~Laemmli, 1970!. Enzyme fractions were pooled and dialyzed over-night against 2 L of 50 mM sodium phosphate pH 7.5 and thenloaded onto a Blue-Sepharose~Sigma! column. The enzyme waseluted with a three column volume salt gradient~0–1 M NaCl!~Sreerama & Sladek, 1993!. Final enzyme purity~estimated to be.90%! was assessed by Coomassie Blue-stained SDS-PAGE~Laemmli, 1970!. The purified enzymes were concentrated usingan Amicon~Beverly, Massachusetts! stirred-cell concentrator andstored at2208C. The final protein concentration was determinedusing a Bio-Rad~Hercules, California! protein assay kit and bo-vine serum albumin as a standard. For the E333Q mutant enzyme,a three column volume EDTA gradient~0–20 mM! in 50 mMsodium phosphate pH 7.5, 500 mM NaCl was employed to elutethe protein from the Chelating-Sepharose column. No enzyme ac-tivity was found in the column fractions containing E333Q, soSDS gels and Western blots were used to locate the enzyme in theeluted column fractions. Partially purified enzyme fractions werepooled and dialyzed against 2 L of 50 mM sodium phosphatepH 7.5, 500 mM NaCl to remove the EDTA. The dialyzed proteinwas concentrated using an Amicon stirred-cell concentrator andstored at2208C.

Fluorescence assays

Activity assays were performed by measuring the rate of increasedfluorescence due to the formation of NADH in 100 mM sodiumphosphate~pH 7.4! at 258C ~Farrés et al., 1994!. Enhancement ofNADH fluorescence when bound to the enzymes followed proce-dures previously described~Wang & Weiner, 1995!.

Spectroscopic assay of esterase activity

Activity assays were performed by measuring the hydrolysis ofNPA at 400 nm in 100 mM sodium phosphate~pH 7.4! at 258C inthe absence or presence of added NAD~Takahashi & Weiner, 1981!.

Western blot analysis of SDS gels

After SDS gel electrophoresis~Laemmli, 1970!, proteins weretransferred to nitrocellulose~Towbin et al., 1979!. Protein bandswere detected using rabbit polyclonal antibodies raised againstrecombinant human stomach ALDH3 and goat antirabbit secondantibody conjugated with alkaline phosphatase. 4-Bromo-5-chloro-indolyl phosphate and nitroblue tetrazolium were used for colordevelopment~Mierendorf et al., 1987!.

Acknowledgments

The authors would like to thank Dr. Alan Townsend, Wake Forest Univer-sity, for the gift of the plasmid containing the cDNA clone for humanstomach ALDH3. We would also like to thank Chris Colbert, Departmentof Biological Sciences, Purdue University, for his assistance in generatingFigures 3 and 4, and Michael Thompson, St. Joseph’s College, for histechnical assistance during a MARC Summer Undergraduate Internship.

This work was supported by a NIH Grant AA05812. This is journal papernumber 15986 from the Purdue Agricultural Experimental Station.

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differences in the roles of conserved glutamic acid residues in the

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