Proc. Natl. Acad. Sci. USAVol. 92, pp. 11666-11670, December 1995Biochemistry

A highly active decarboxylating dehydrogenase with rationallyinverted coenzyme specificityRIDONG CHEN, ANN GREER, AND ANTONY M. DEANDepartment of Biological Chemistry, The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064

Communicated by Daniel E. Koshland, Jr., University of California, Berkeley, CA, September 1, 1995

ABSTRACT The isocitrate dehydrogenase of Escherichiacoli, which lacks the Rossmann fold common to other dehy-drogenases, displays a 7000-fold preference for NADP overNAD (calculated as the ratio of kcat/Km). Guided by x-raycrystal structures and molecular modeling, site-directed mu-tagenesis has been used to introduce six substitutions in theadenosine binding pocket that systematically shift coenzymepreference toward NAD. The engineered enzyme displays an850-fold preference for NAD over NADP, which exceeds the140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only oneis identical in all related NAD-dependent enzyme sequences-strict adherence to homology as a criterion for replacing theseamino acids impairs function. Two additional mutations atremote sites improve performance further, resulting in a finalmutant enzyme with kinetic characteristics and coenzymepreference comparable to naturally occurring homologousNAD-dependent enzymes.

Descriptions of the determinants of specificity based on pro-tein structures represent plausible hypotheses that beg exper-imental verification. A thorough understanding of the deter-minants of specificity is demonstrated whenever the prefer-ence for two substrates is inverted by rational means.Dehydrogenases discriminate among nicotinamide coenzymesthrough interactions established between the protein and the2'-phosphate of NADP and the 2'- and 3'-hydroxyls of NAD.Engineering dihydrolipoamide and malate dehydrogenasesdemonstrates that changing the preference of an NAD-dependent enzyme can be achieved by introducing positivelycharged residues to neutralize the negatively charged 2'-phosphate of NADP (1, 2). Yet, as earlier attempts to invertthe preference of glutathione reductase and glutamate dehy-drogenase illustrate, engineering the preference of an NADP-dependent enzyme toward NAD is more troublesome (3, 24).Perhaps, the strict reliance on homology as a criterion forreplacing amino acids is insufficient to optimize directionalinteractions, such as hydrogen bonds to the 2'- and 3'-hydroxyls of NAD.The decarboxylating dehydrogenases, of which Escherichia

coli isocitrate dehydrogenase (IDH) and Thermus thermophilusisopropylmalate dehydrogenase (IMDH) are members, forman ancient family of dehydrogenases sharing 25% amino acidsequence identity and a common catalytic mechanism (4, 5).They also share a common protein fold (Fig. 1A), one that istopologically distinct from other dehydrogenases of knownstructure and that lacks the afc3ap binding motif characteristicof the nucleotide binding Rossmann fold. Instead, the aden-osine moiety of coenzyme binds in a pocket constructed fromtwo loops and an a-helix in IDH, although the latter issubstituted by a (3-turn in IMDH (Fig. 1A) (4, 5).

Specificity in E. coli IDH is conferred by interactions amongArg-395, Tyr-345, Tyr-391, and Arg-292' with the 2'-

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

phosphate of bound NADP (Fig. 1B; E. coli IDH numbering).These residues are conserved in prokaryotic NADP-de-pendent IDHs and replaced with a variety of residues in theNAD-dependent dehydrogenases (Table 1). In T. thermophilusIMDH, there is no site equivalent to position 395, whilereplacements Ser-292', Ile-345, and Pro-391 eliminate allfavorable interactions with the 2'-phosphate (Fig. 1B). Spec-ificity in IMDH is conferred by the conserved Asp-344, whichforms a double hydrogen bond with the 2'- and 3'-hydroxyls ofthe adenosine ribose of NAD, shifting its position and perhapschanging the ribose pucker from C3'-endo to C2'-endo. Notonly are these movements incompatible with the strong 2'-phosphate interactions seen in IDH but also the negativecharge on Asp-344 may repel NADP. Indeed, the dramaticdrop in the specificity of E. coli IDH toward isocitrate uponphosphorylation of an active site Ser is caused by electrostaticrepulsion of the y-carboxylate of its carboxylic acid substrate(10, 11).

Herein, guided by a knowledge of the determinants ofcoenzyme specificity and molecular modeling, we engineer ahighly active NAD-specific enzyme in the nucleotide bindingdomain of the NADP-dependent IDH of E. coli (5). Only oneof the six substitutions introduced around the binding pocketare identical in all NAD-dependent IMDHs.

MATERIALS AND METHODSSite-Directed Mutagenesis. Plasmid pTK513, which carries

the kcd gene inserted into pEMBL18- (12), was used togenerate uridine-labeled template in E. coli CJ236 with helperphage R401. Oligonucleotide primers containing the necessarymismatches were synthesized on a Biosearch model 8700 DNAsynthesizer and were used to introduce mutations into the kcdgene by the Kunkel method (6) with a kit from Bio-Rad.Putative mutants were screened by kinetic analysis and thenconfirmed by dideoxynucleotide sequencing (13).

Cell Growth and Enzyme Purification. After transforma-tion of the mutated plasmids into E. coli SL4 (AIDH), cultureswere grown to full density overnight in 100 ml of Luria brothat 37°C in the presence of ampicillin (60 gg/ml). Purificationof the enzymes, by the procedure of Garnak and Reeves (14)as modified by Dean and Koshland (15), involves ammoniumsulfate precipitation, DEAE anion chromatography, and af-finity chromatography using Affi-Gel Blue. All preparationsare 95% free of contaminating enzyme, as judged by Coomas-sie blue staining after SDS/PAGE electrophoresis.

Kinetic Analyses. The kinetics of IDHs were determined inKAC buffer (25 mM Mops/100mM NaCl/5 mM MgCl2/1 mMdithiothreitol, pH 7.5) at 21°C in the presence of 1 mMDL-isocitrate (10). Data were collected on a Hewlett-Packardmodel 8452A single-beam diode-array spectrophotometer.The rates of reaction were determined by monitoring theproduction ofNAD(P)H at 340 nm in a 1-cm light path by usinga molar extinction coefficient of 6200 M-1cm-1. Protein

Abbreviations: IDH, isocitrate dehydrogenase; IMDH, isopropyl-malate dehydrogenase.

11 tt

Dow

nloa

ded

by g

uest

on

Mar

ch 1

0, 2

021

Proc. Natl. Acad. Sci. USA 92 (1995) 11667

FIG. 1. (A) Ribbon trace of a monomer of E. coli IDH (,B-sheets are green, a-helices are purple, and loops are white), showing the positionsof the six Cys residues (numbered left to right are Cys-405, -332, -127, -301, and -201 with Cys-194 in the lower loop: all marked by yellow sidechains). The large cleft between the two domains contains the active site marked by isocitrate and NADP (carbon is white, nitrogen is blue, oxygenis red, and phosphorous is light yellow). (B) Superposition of the cofactor binding pockets of the NADP-dependent E. coli IDH (5, 6) with twowaters (red spheres) and the NAD-dependent T. thermophilus IMDH (ref. 4; yellow). Side chains of Ile-37, Val-41, Ile-320, His-339, Ala-342, andVal-351, the aliphatic portion of the side chains of Asn-352 and Asp-392, and the main-chain residues Gly-321 and Asn-352 form the binding pocket(E. coli IDH numbering). All residues are identical in T thernophilus IMDH except for conservative substitutions replacing Ile-320 with Leu andVal-351 with Ala. N2 and N6, common to the adenosine 2',3'-bisphosphate moiety of NADP, form hydrogen bonds to the main-chain amide andcarbonyl of Asn-352. A dipole-quadrupole interaction between the adenine N6 and the His ring is evident in IMDH, but the low pH conditionsnecessary for crystallization of IDH may have disrupted this interaction. Specificity in IDH is conferred by interactions among residues Arg-292'(on the second domain of the second subunit), Arg-395, Tyr-345, and Tyr-391 with the 2'-phosphate of bound NADP. Specificity in IMDH isconferred by Asp-344, which forms a double hydrogen bond with the 2'- and 3'-hydroxyls of the adenosine ribose of NAD and may also repel the2'-phosphate of NADP.

concentrations were determined at 280 nm by using a molarextinction coefficient of 66,330 M-1-cm-1 (15). Nonlinear leastsquares Gauss-Newton regressions were used to determine thefit of the data to the Michaelis-Menten model.

Molecular Modeling. Molecular modeling was conducted ona Silicon Graphics 4D120/GTX using QUANTA/CHARMM soft-ware program and visualized using the Crystal Eyes stereoviewing system. X-ray crystallographic structures of the binarycomplexes of IDH with NADP and IMDH with NAD weresuperimposed by least squares minimization of main-chainatoms surrounding the nucleotide binding pockets. Amino acidsubsitutions were modeled assuming that the polypeptidebackbone remained unchanged, and side chains were adjustedby rotating torsional bonds to establish favorable interactionswith NAD.

RESULTSSubstitutions in the Coenzyme Binding Pocket. Site-

directed mutagenesis was used to replace Lys-344 with Asp andTyr-345 with Ile. Both Asp-344 and Ile-345 are identical in allknown prokaryotic NAD-dependent decarboxylating dehydro-genases (Table 1). As expected from a loss of hydrogenbonding and the introduction of a potentially repulsive aspar-

tate, the performance with NADP was greatly reduced (Table2). Although preference no longer favored NADP, the per-formance with NAD suggests that no new interactions wereestablished with this coenzyme.

Val-351 ofIDH is either conserved or replaced by Ala in theNAD-dependent enzymes (Table 1). Modeling suggested thatthe reduced bulk of Ala might allow the adenosine to shift,bringing the 2'- and 3'-hydroxyls of the attached ribose closerto Asp-344. Site-directed mutagenesis was used to generate thetriple mutant with striking results. The 14-fold increase inperformance with NAD was consonant with the formation ofa hydrogen bond. Preference now favored NAD by a factor of4 (Table 2).

Tyr-391 is replaced with Pro in T. thermophilus IMDH (Fig.1B). This substitution removes a hydrogen bond to the 2'-phosphate and alters the local secondary structure froma-helix to 3-turn. Pro, common to many IMDHs, was notintroduced at site 391 to avoid disrupting the a-helix of IDH.Nor was Phe chosen because it might become buried in thehydrophobic pocket, thereby hindering the approach of theadenosine of NAD toward Asp-344. Sequence alignmentssuggested that either Gly or Arg might be introduced at thissite. Further along the a-helix of IDH is Arg-395, which alsohydrogen bonds to the 2'-phosphate, but which has no equiv-

Biochemistry: Chen et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

0, 2

021

Proc. Natl. Acad. Sci. USA 92 (1995)

Table 1. Alignments of the primary sequences of the decarboxylating dehydrogenases around the adenosine binding pocket

Enzyme Sequence

NADP-dependent IDHE. coliT. thermophilusVibrio sp.

NAD-dependent IMDHT. thermophilusT. aquaticus

E. coliS. typhimuriumB. subtilisL. interrogansA. tumefaciensY lipolyticaS. cerevisiae

C. utilisS. pombe

NAD-dependent IDHS. cerevisiae

NAD-dependent TDHP. putida

283 294DAFLQQILLRPAEYDNAAHQLVKRPEQFDAMLQQVLLRPAEY

DAMAMHLVRSPARFDAMAMHLVKNPARFDNATMQLIKDPSQFDNATMQLIKDPSQFDNAAMQLIYAPNQFDNAAMQLIVNPKQFDAGGMQLVRKPKQFDSAAMILIKQPSKMDSAAMILVKNPTHLDSAAMILIKYPTQLDSAAMLLVKSPRTL

DNSVLKVVTNPSAY

DILCARFVLQPERF

** * *

316 325QVGGIGIAPGANLIGGLGFAPSANQVGGIGIAPGAN

LPGSLGLLPSASLPGSLGLLPSASITGSMGMLPSASITGSMGMLPSASLTGSLGMLPSASITGSIGMLPSASLTGSLGMLPSASIPGSLGLLPSASIPGSLGLLPSASIPGSLGLLPSASIPGSLGLLPSAS

SAGSLGLTPSAN

CAGTIGIAPSAN

** *

334 344 351 357LFEATHGTAPKYAGQDKVNPGSIILSIFEAVHGSAPKYAGKNVINPTAVLLSVFZATHGTAPKYAGKNKVNPGSVILS

VFEPVHGSAPDIAGKGIANPTAAILSVFZPVHGSAPDIAGKGIANPTAAILSLYZPAGGSAPDIAGKNIANPIAQILSLYEPAGGSAPDIAGKNIANPIAQILSLFZPVHGSAPDIAGKGMANPFAAILSLYEPSGGSAPDIAGKGVANPIAQVLSMYEPVHGSAPDIAGKSIANPIAMIASLYEPCHGSAPDL.GKQKVNPIATILSLYEPCHGSAPDL.PKNKVNPIATILSLYEPCHGSAPDL.PANKVNPIATILSLVEPIHGSAPDIAGKGIVNPVGTILS

IFZAVHGSAPDIAGQKDANPTALLLS

LFEPVHGSAPDIFGKNIANPIAMIWS

IDH numbering is used throughout, asterisks denote sites subjected to mutagenesis, and boldface type denotes rigidly conserved amino acids.IDH was from E. coli, T. thermophilus, and Saccharomyces cerevisiae (7), and Vibrio sp. (8); IMDH was from T-. thermophilus, Thermus aquaticus,E. coli, Bacillus subtilis, Leptospira interrogans, Agrobacterium tumefaciens, Yarrowia lipolytica, Saccharomyces cerevisiae, Candida utilis, Schizosac-charomyces pombe, and Salmonella typhimurium (7). TDH, tartrate dehydrogenase (9). P. putida, Pseudomonas putida.

alent in the (3-turn of IMDH. If sequence alignments suggestthat Arg occupies this "site" in several IMDHs, the secondarystructure of the (3-turn in IMDH shows that the side chain willpoint away from the nucleotide binding site. Sequence align-ments suggested the Arg might be replaced with Gly or Ala.The introduction of Gly residues at both sites improved

preference by destroying activity with NADP (no catalysis wasdetectable). However, the performance with NAD was re-duced below that of the wild-type enzyme (K344D/Y345I/V351A/Y391G/R395G had a Km of 1400 pLM, a kcat of 0.284sec-1, and a performance kcat/Km of 0.0002 ,uM-1lsec-1). Asecond mutant (K344D/Y345I/V351A/Y391R/R395A) pro-duced comparable results. Instead, Tyr-391 was replaced byhydrophilic Lys that, being shorter than the Arg found in manyIMDHs, was less likely to interact the 2'-phosphate of NADP.Although at the surface, modeling indicated that replacing

Arg-395 in the a-helix with Val, Thr, Leu, Met, or Ile mightgenerate steric effects with adjacent residues. Hence, Ser, apolar residue suitable for replacements at the surface of aprotein, was chosen to replace Arg-395. The introduction ofY391K and R395S to generate K344D/Y345I/V351A/Y391K/R395S caused a dramatic decrease in performancewith NADP, as expected from the loss of two hydrogen bondsto the 2'-phosphate and a modest increase in performance withNAD (Table 2). The preference forNAD over NADP was thus100-fold.A shift in the loop formed by residues 316-322 alters the

orientation of Leu-320 in IMDH, nudging the adenosinemoiety of NAD toward Arg-344. This shift, generated byPro-317, is stabilized by a hydrogen bond between Asp-392 andthe hydroxyl of Ser-319 that precisely replaces by a boundwater with a similar function in IDH. All attempts to engineer

Table 2. Kinetic parameters of wild-type and mutant enzymes toward NADP and NAD

NADP NAD

Performance, Performance Preference,Km, kcat, kcat/Km, Km, kcat, kcat/Km, NADP performance

Enzyme ,uM sec- 1 M-1 . sec-I1 uM sec- 1 .&M-1-sec-1 /NAD performanceE. coli IDH at 21°CabcdefghKYVYRRCC (wild type) 17 80.5 4.7 4700.0 3.22 0.00069 6900DI------ 7,300 6.3 0.00086 3300.0 2.59 0.00078 1.1DIA----- 6,400 18.0 0.0028 850.0 9.39 0.011 0.25DIAKS--- 11,300 2.0 0.00018 290.0 6.00 0.021 0.009DIAKSD-- 32,200 0.39 0.000012 924.0 9.74 0.011 0.001DIAKS-Y- 2,800 3.34 0.0012 108.0 11.4 0.106 0.011DIAKS-YI 5,800 4.70 0.00081 99.0 16.20 0.164 0.005

Saccharomyces cerevisiaeWild-type IDH at 24°C (16, 17) 210 40 0.190Wild-type IMDH at 30°C (18) 140 14.48 0.103

Salmonella typhimuriumWild-type IMDH at 24°C (19) 100 31.5 0.315

T. thermophilusWild-type IMDH at 65°C (7) 12,300 29.9 0.00243 40 13.6 0.34 0.007All apparent standard errors are <15% of the estimates. For E. coli IDH, residues at the following positions are shown: a, 344; b, 345; c, 351;

d, 391; e, 395; f, 292; g, 332; h, 201. Dashes indicate wild-type residues.

* *

338 395TVTYDFERLMVLTGDVVGYDTVTYDFERLM

TPPPDLGGSATPPPDLGGSAIRTGDLARGAVRTGDLARGAKRTRDLARSEKRTRDIEVGSIRTADIMADGITTADIGGSSIRTGDLGGSNIRTGDLKGTNLYTRDLGGEA

NRTGDLAGTA

SVTPDMGGTL

11668 Biochemistry: Chen et aL

Dow

nloa

ded

by g

uest

on

Mar

ch 1

0, 2

021

Proc. Natl. Acad. Sci. USA 92 (1995) 11669

the loop in IDH (e.g., Q316L, V317P, G319S, and/or I320L)reduced performance toward both coenzymes. Engineeringthe entire loop produced similar results (the performance ofK344D/Y3451/V351A/Y391K/R395S/Q316L/V317P/G319S/1320L was 0.00096 uM- lsec- 1 with NAD and 0.00001AM-1 sec-1 with NADP). All substitutions in the loop wereomitted from the next round of engineering.

Arg-292' forms a hydrogen bond from the small domain ofthe second subunit to the 2'-phosphate of NADP and isreplaced by Ser in T. thermophilus IMDH, and a wide varietyof amino acids in other NAD-dependent enzymes (Table 1).The introduction of Asp improved the preference for NADover NADP to 850-fold, exceeding the 140-fold preferencedisplayed by T. thermophilus IMDH (Table 2). However, thissubstitution also increased the Km ofNAD by a factor of 3 andwas, therefore, omitted from the next round of engineering.

Substitutions Outside the Coenzyme Binding Pocket. Asjudged by improved performance with NAD by a factor of 30,K344D/Y345I/V351A/Y391K/R395S was the most success-ful mutant so far generated. Nevertheless, it was far less activethan natural homologous NAD-dependent decarboxylatingdehydrogenases (Table 2). We decided to investigate thepossibility that amino acid substitutions outside the nucleotidebinding pocket might promote binding and catalysis. Six Cysresidues were targeted because they are limited in number anddistributed haphazardly with respect to the coenzyme bindingpocket: Cys-405 is immediately adjacent, Cys-332 lies on a(3-sheet that traverses the pocket, Cys-127 supports Arg-159,which hydrogen bonds to the a-carboxylate of isocitrate,Cys-301 lies deep in the hydrophobic core of the seconddomain, Cys-201 lies adjacent to a loop that forms part of theactive site ip the dimer, and Cys-194 lies at the dimer interfacein the extended loop (Fig. 1A). Amino acid substitutions wereintroduced by using degenerate oligonucleotides, one encod-ing bulkier residues (Phe, Tyr, and His) and the other encodinglonger residues (Ile, Lys, and Met). In accommodating thesebulky residues, subtle conformational shifts might be trans-mitted into the adenosine binding pocket and the active site,fortuitously improving performance.

Molecular modeling suggested that substitutions at Cys-127would disturb isocitrate binding by displacing Arg-159, those atthe deeply buried Cys-301 would disrupt the hydrophobic core,while those at Cys-194 would interfere with subunit interac-tions. Indeed, substitutions at these three sites greatly reducedperformance of the K344D/Y345I/V351A/Y391K/R395Smutant by at least a factor of 100 (data not shown). AlthoughCys-405 is adjacent to the adenosine binding pocket, allsubstitutions were predicted to have negligible structural ef-fects because minor torsion changes allow side chains to beexposed to solvent. As expected, none of the substitutionsintroduced influenced enzyme performance, which in all casesremained comparable to the parent K344D/Y345I/V351A/Y391K/R395S mutant (Table 2 and data not shown).The partially buried Cys-332 in the (3-sheet that traverses the

adenosine binding pocket is also adjacent to active site resi-dues. Replacements with Phe, and especially Tyr, increasedkcat by 2-fold and decreasedKm by 3-fold (Table 2). Cys-201 liesadjacent to the super secondary structure containing an activesite Lys-230'. Replacement with Ile caused a modest improve-ment in kcat (Table 2). All bulkier residues impaired perfor-mance by factors >100 (data not shown).

DISCUSSIONPrevious studies reveal that using homology as a strict guide fordetermining the introduction of amino acid substitutions suc-ceeds as often as it fails. For example, the replacement of asingle Glu with Arg converts Bacillus stearothermophilus lac-tate dehydrogenase into a highly specific malate dehydroge-nase (20), while the replacement of Arg with Gln in E. coli

malate dehydrogenase merely generates an enzyme with poorsubstrate specificity (21). Similarly, introducing positivelycharged side chains into the Rossmann fold of the E. coliNAD-dependent dihydrolipoamide dehydrogenase generatesa highly specific NADP-dependent enzyme (1), while substi-tutions in the Rossmann fold of the NADP-dependent E. coliglutathione reductase fail to generate an enzyme with a strictpreference for NAD, even at optimal pH (3). Indeed, engi-neering IDH performance is frequently impaired when ho-mology is the sole criterion governing the introduction ofresidues. For example, the substitution of Gly at sites 391 and395 reduces performance with NAD by a factor of 55, whileengineering the loop between Gln-316 and Gly-321 reducesperformance with both coenzymes by factors >10.

In this study three approaches, substitutions based on ho-mology, alternative substitutions within binding sites, andsubstitutions outside binding sites, were combined to invert thecoenzyme preference of E. coli IDH while retaining perfor-mance.

Substitutions Based on Homology. The first two mutations,K344D and Y345I, were based on homology. Sequence align-ments indicated that Asp-334 is rigidly conserved in all relatedNAD-dependent enzymes, and structural analysis indicated

1.2a. 10.-.

s 0.8

3r 3:O 0 0.6

0.4

o- 0IL 0.210

_ 200<z a

3-= 150

31000Io

50

F.

D-- 1 UUUz 100

< 10z

0

0.1

CL 0.01

0.001

100Q HX C) >4 >d> cnuuv

', H H H H H

FIG. 2. Systematic shift in coenzyme preference generated inengineered mutants of IDH. Differences in the height of the histogramcolumns represent the degree to which performance (kcat/Km) andpreference [calculated as the ratio (kcat/Km)NADp/(kcat/Km)NAD] isshifted by each set of mutations (denoted by the single-letter aminoacid code).

i

;

Biochemistry: Chen et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

0, 2

021

Proc. Natl. Acad. Sci. USA 92 (1995)

that it hydrogen bonds to the 2'- and 3'-hydroxyls of theadenosine ribose of NAD while potentially repelling the2'-phosphate of NADP. Structural analysis also revealed thatthe substitution Y3451, a conserved residue in many relatedNAD-dependent enzymes, not only removes a hydrogen bondto the 2'-phosphate of NADP but might eliminate any sterichindrance preventing the formation of the hydrogen bondsbetween the ribose hydroxyls and Asp-334. Nevertheless, theresulting double mutant showed no improved performancewith NAD (Fig. 2).

Substitutions Based on Design. Structural analysis sug-gested that the bulk of Val-351 might prevent the adenosineshifting, thereby preventing Asp-344 from forming hydrogenbonds to 2'- and 3'-hydroxyls of the adenosine ribose of NAD.Replacement with Ala, found in some but not all NAD-dependent enzymes, produced the desired effect: the triplemutant had a 14-fold increase in performance with, and a4-fold preference for, NAD (Fig. 2). The next two substitutionsintroduced were Y391K and R395S. The criteria for introduc-ing these substitutions were removal of hydrogen bonds to the2'-phosphate of NADP, maintenance of local secondary struc-ture, avoidance of steric effects hindering the approach of theadenosine ribose toward Asp-344, and hydrophilicity, thesebeing surface residues. The 2-fold increase in performance and30-fold increase in preference with NAD (Fig. 2) was achievedsolely by rational design since these sites have no directcounterparts in IMDH, where the a-helix is replaced by a,-turn.

Substitutions Outside the Coenzyme Binding Pocket. Ad-ditional mutations in the binding pocket impaired perfor-mance with NAD. Furthermore, the failure of several otherstudies to invert preference based on homology or the use ofalternative' substitutions within binding sites, suggested thepossibility that substitutions outside the binding site mightimprove performance with NAD.The choice of replacing the six Cys residues (Fig. 1A) was

arbitrary, save their limited number and haphazard distribu-tion with respect to the coenzyme binding site. The degenerateoligonucleotides encoded larger amino acids to force changesin conformation. A net 8-fold increase in performance wasgenerated by substitutions at two of the six sites. The mech-anisms by which this improvement was generated awaitsfurther structural analysis. However, the implication is that thecombined effects of many substitutions outside binding sitesand catalytic enters may be considerable in aggregate. Theseobservations may help explain both the general difficultyencountered in engineering enzyme function and the reasonwhy enzymes are so large.

Kinetic Properties of the Final Mutant. Seven amino acidsubstitutions introduced into wild-type E. coli IDH cause ashift in preference from NADP to NAD by a factor >106:performance with NADP was reduced 6000-fold, and perfor-mance with NAD increased 240-fold (Fig. 2). Of the sevensuccessful substitutions introduced into IDH, only two werebased on strict homology with the NAD-dependent enzymes(Table 1). Our final mutant displays a preference for NADover NADP of 200-fold; has an NAD Michaelis constant of 100,tM, representing an improvement by a factor of 50, and a kcatof 16.2 sec-1, which represents an increase by a factor of 5(Table 2). The results suggest that a hydrogen bond betweenthe adenosine ribose ofNAD and Asp-344, as seen in the x-raystructure of the IMDH binary complex, may have beensuccessfully established. Note that the Michaelis constant ofisocitrate remains unchanged at 10 ,uM, suggesting that thesemutations have no effect on substrate binding.The kinetic characteristics of the final engineered enzyme

compare favorably with natural NAD-dependent IDHs and

IMDHs from various sources (Table 2). The fact that theMichaelis constant of the mutant enzyme is higher towardNAD than that of the wild-type enzyme toward NADP appearstypical of natural IDHs: the Michaelis constants of NAD-dependent enzymes range from 150 ,uM to 800 j,M, whereasthose of the NADP-dependent enzymes range from 2 ,M to20 ,uM (22). Perhaps the strong electrostatic interactions withthe 2'-phosphate of NADP, evident in the crystal structure ofE. coli IDH with NADP (Fig. 1B), are responsible for thisdifference. The reason why the maximum rate of catalysisdisplayed by wild-type IDH utilizing NADP is 5-fold higherthan with the engineered mutant (Table 2) is unclear. Con-formational changes induced by NADP binding in IDH arerestricted to side-chain movements in the immediate vicinity ofthe coenzyme binding pocket (5). The structure of a pseudo-Michaelis ternary complex with NADP and isocitrate has beendetermined (23), but again, no obvious changes in the activesite can be ascribed to changes induced in the coenzymebinding pocket, which lies some 14 A away. Hence, our finalmutant is probably as efficient an NAD-dependent IDH as iscurrently possible to design.

We thank Eric Walters, Jim Hurley, and Bob Kemp for theirthoughtful suggestions. This work was supported by Public HealthService Grant GM-48735 from the National Institutes of Health.

1. Bocanegra, J. A., Scrutton, N. S. & Perham, R. N. (1993) Bio-chemistry 32, 2737-2740.

2. Nishiyama, M., Birktoff, J. J. & Beppu, T. (1993) J. Bio. Chem.268, 4656-4660.

3. Scrutton, N. S., Berry, A. & Perham, R. N. (1990) Nature (Lon-don) 343, 38-43.

4. Hurley, J. H. & Dean, A. M. (1994) Structure 2, 1007-1016.5. Hurley, J. H., Dean, A. M., Koshland, D. E., Jr., & Stroud, R. M.

(1991) Biochemistry 30, 8671-8678.6. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) Methods

Enzymol. 154, 367-382.7. Miyazaki, K. & Oshima, T. (1994) Protein Eng. 7, 401-403.8. Ishii, A. (1993) J. Bacteriol. 175, 6873-6880.9. Tipton, P. A. & Beecher, B. S. (1994) Arch. Biochem. Biophys.

313, 15-21.10. Dean, A. M. & Koshland, D. E., Jr. (1990) Science 249, 1044-

1046.11. Hurley, J. H., Dean, A. M., Sohl, J. L., Koshland, D. E., Jr., &

Stroud, R. M. (1990) Science 249, 1012-1016.12. Thorsness, P. E. & Koshland, D. E., Jr. (1987) J. Biol. Chem. 262,

10422-10425.13. Sanger, F., Nicklen, S. & Coulsen, R. (1977) Proc. Natl. Acad. Sci.

USA 74, 5463-5467.14. Garnak, M. & Reeves, H. C. (1979) J. Biol. Chem. 254, 7915-

7920.15. Dean, A. M. & Koshland, D. E., Jr. (1993) Biochemistry 32,

9302-9309.16. Barnes, L. D., Kuehn, G. D. & Atkinson, D. E. (1971) Biochem-

istry 10, 3939-3945.17. Cupp, J. R. & McAlister-Henn, L. (1993) Biochemistry 32, 9323-

9328.18. Hsu, Y.-P. & Kolhaw, G. B. (1980)J. Biol. Chem. 255,7255-7260.19. Parsons, S. J. & Burns, R. 0. (1970) Methods Enzymol. 17A,

793-799.20. Wilks, H. M., Hart, K. W., Feeney, R., Dunn, C. R., Muirhead,

H., Chia, W. N., Barstow, D. A., Atkinson, T., Clarke, A. R. &Holbrook, J. J. (1988) Science 242, 1541-1544.

21. Nicholls, D. J., Miller, J., Scawen, M. D., Clarke, A. R., Hol-brook, J. J., Atkinson, T. & Goward, C. R. (1992) Biochem.Biophys. Res. Commun. 189, 1057-1062.

22. Chen, R. & Gadal, P. (1990) Plant Physiol. Biochem. 28, 411-418.23. Stoddard, B. L., Dean, A. M. & Koshland, D. E., Jr. (1993)

Biochemistry 32, 9310-9316.24. Haeffner-Gormley, L., Chen, Z., Zalkin, H. & Colman, R. (1992)

Biochemistry 31, 7807-7814.

11670 Biochemistry: Chen et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

0, 2

021