The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehy- drogenases, displays a 7000-fold preference for NADP ...
Proc. Natl. Acad. Sci. USA
Vol. 92, pp. 11666-11670, December 1995 Biochemistry
A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity RIDONG CHEN, ANN GREER, AND ANTONY M. DEAN Department 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
phosphate of bound NADP (Fig. 1B; E. coli IDH numbering). These residues are conserved in prokaryotic NADP-dependent IDHs and replaced with a variety of residues in the NAD-dependent dehydrogenases (Table 1). In T. thermophilus IMDH, there is no site equivalent to position 395, while replacements Ser-292', Ile-345, and Pro-391 eliminate all favorable interactions with the 2'-phosphate (Fig. 1B). Specificity in IMDH is conferred by the conserved Asp-344, which forms a double hydrogen bond with the 2'- and 3'-hydroxyls of the adenosine ribose of NAD, shifting its position and perhaps changing the ribose pucker from C3'-endo to C2'-endo. Not only are these movements incompatible with the strong 2'phosphate interactions seen in IDH but also the negative charge on Asp-344 may repel NADP. Indeed, the dramatic drop in the specificity of E. coli IDH toward isocitrate upon phosphorylation of an active site Ser is caused by electrostatic repulsion of the y-carboxylate of its carboxylic acid substrate (10, 11). Herein, guided by a knowledge of the determinants of coenzyme specificity and molecular modeling, we engineer a highly active NAD-specific enzyme in the nucleotide binding domain of the NADP-dependent IDH of E. coli (5). Only one of the six substitutions introduced around the binding pocket are identical in all NAD-dependent IMDHs.
The isocitrate dehydrogenase of Escherichia ABSTRACT coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NADdependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequencesstrict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.
Descriptions of the determinants of specificity based on protein structures represent plausible hypotheses that beg experimental verification. A thorough understanding of the determinants of specificity is demonstrated whenever the preference for two substrates is inverted by rational means. Dehydrogenases discriminate among nicotinamide coenzymes through interactions established between the protein and the 2'-phosphate of NADP and the 2'- and 3'-hydroxyls of NAD. Engineering dihydrolipoamide and malate dehydrogenases demonstrates that changing the preference of an NADdependent enzyme can be achieved by introducing positively charged residues to neutralize the negatively charged 2'phosphate of NADP (1, 2). Yet, as earlier attempts to invert the preference of glutathione reductase and glutamate dehydrogenase illustrate, engineering the preference of an NADPdependent enzyme toward NAD is more troublesome (3, 24). Perhaps, the strict reliance on homology as a criterion for replacing amino acids is insufficient to optimize directional interactions, such as hydrogen bonds to the 2'- and 3'hydroxyls of NAD. The decarboxylating dehydrogenases, of which Escherichia coli isocitrate dehydrogenase (IDH) and Thermus thermophilus isopropylmalate dehydrogenase (IMDH) are members, form an ancient family of dehydrogenases sharing 25% amino acid sequence identity and a common catalytic mechanism (4, 5). They also share a common protein fold (Fig. 1A), one that is topologically distinct from other dehydrogenases of known structure and that lacks the afc3ap binding motif characteristic of the nucleotide binding Rossmann fold. Instead, the adenosine moiety of coenzyme binds in a pocket constructed from two loops and an a-helix in IDH, although the latter is substituted by a (3-turn in IMDH (Fig. 1A) (4, 5). Specificity in E. coli IDH is conferred by interactions among Arg-395, Tyr-345, Tyr-391, and Arg-292' with the 2'-
MATERIALS AND METHODS Site-Directed Mutagenesis. Plasmid pTK513, which carries the kcd gene inserted into pEMBL18- (12), was used to generate uridine-labeled template in E. coli CJ236 with helper phage R401. Oligonucleotide primers containing the necessary mismatches were synthesized on a Biosearch model 8700 DNA synthesizer and were used to introduce mutations into the kcd gene by the Kunkel method (6) with a kit from Bio-Rad. Putative mutants were screened by kinetic analysis and then confirmed by dideoxynucleotide sequencing (13). Cell Growth and Enzyme Purification. After transformation of the mutated plasmids into E. coli SL4 (AIDH), cultures were grown to full density overnight in 100 ml of Luria broth at 37°C in the presence of ampicillin (60 gg/ml). Purification of the enzymes, by the procedure of Garnak and Reeves (14) as modified by Dean and Koshland (15), involves ammonium sulfate precipitation, DEAE anion chromatography, and affinity chromatography using Affi-Gel Blue. All preparations are 95% free of contaminating enzyme, as judged by Coomassie blue staining after SDS/PAGE electrophoresis. Kinetic Analyses. The kinetics of IDHs were determined in KAC buffer (25 mM Mops/100 mM NaCl/5 mM MgCl2/1 mM dithiothreitol, pH 7.5) at 21°C in the presence of 1 mM DL-isocitrate (10). Data were collected on a Hewlett-Packard model 8452A single-beam diode-array spectrophotometer. The rates of reaction were determined by monitoring the production of NAD(P)H at 340 nm in a 1-cm light path by using a molar extinction coefficient of 6200 M-1cm-1. Protein
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Abbreviations: IDH, isocitrate dehydrogenase; IMDH, isopropylmalate dehydrogenase. 11 tt
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Proc. Natl. Acad. Sci. USA 92 (1995)
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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 positions of 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 side chains). The large cleft between the two domains contains the active site marked by isocitrate and NADP (carbon is white, nitrogen is blue, oxygen is red, and phosphorous is light yellow). (B) Superposition of the cofactor binding pockets of the NADP-dependent E. coli IDH (5, 6) with two waters (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, and Val-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 and Val-351 with Ala. N2 and N6, common to the adenosine 2',3'-bisphosphate moiety of NADP, form hydrogen bonds to the main-chain amide and carbonyl of Asn-352. A dipole-quadrupole interaction between the adenine N6 and the His ring is evident in IMDH, but the low pH conditions necessary 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 is conferred 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 the 2'-phosphate of NADP.
concentrations were determined at 280 nm by using a molar extinction coefficient of 66,330 M-1-cm-1 (15). Nonlinear least squares Gauss-Newton regressions were used to determine the fit of the data to the Michaelis-Menten model. Molecular Modeling. Molecular modeling was conducted on a Silicon Graphics 4D120/GTX using QUANTA/CHARMM software program and visualized using the Crystal Eyes stereo viewing system. X-ray crystallographic structures of the binary complexes of IDH with NADP and IMDH with NAD were superimposed by least squares minimization of main-chain atoms surrounding the nucleotide binding pockets. Amino acid subsitutions were modeled assuming that the polypeptide backbone remained unchanged, and side chains were adjusted by rotating torsional bonds to establish favorable interactions with NAD.
RESULTS Substitutions in the Coenzyme Binding Pocket. Sitedirected mutagenesis was used to replace Lys-344 with Asp and Tyr-345 with Ile. Both Asp-344 and Ile-345 are identical in all known prokaryotic NAD-dependent decarboxylating dehydrogenases (Table 1). As expected from a loss of hydrogen bonding and the introduction of a potentially repulsive aspar-
tate, the performance with NADP was greatly reduced (Table 2). Although preference no longer favored NADP, the performance with NAD suggests that no new interactions were established with this coenzyme. Val-351 of IDH is either conserved or replaced by Ala in the NAD-dependent enzymes (Table 1). Modeling suggested that the reduced bulk of Ala might allow the adenosine to shift, bringing the 2'- and 3'-hydroxyls of the attached ribose closer to Asp-344. Site-directed mutagenesis was used to generate the triple mutant with striking results. The 14-fold increase in performance with NAD was consonant with the formation of a hydrogen bond. Preference now favored NAD by a factor of 4 (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 from a-helix to 3-turn. Pro, common to many IMDHs, was not introduced at site 391 to avoid disrupting the a-helix of IDH. Nor was Phe chosen because it might become buried in the hydrophobic pocket, thereby hindering the approach of the adenosine of NAD toward Asp-344. Sequence alignments suggested that either Gly or Arg might be introduced at this site. Further along the a-helix of IDH is Arg-395, which also hydrogen bonds to the 2'-phosphate, but which has no equiv-
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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 IDH E. coli T. thermophilus Vibrio sp. NAD-dependent IMDH T. thermophilus T. aquaticus E. coli S. typhimurium B. subtilis L. interrogans A. tumefaciens Y lipolytica S.
cerevisiae
C. utilis S. pombe NAD-dependent IDH S.
cerevisiae
*
*
**
283 294 DAFLQQILLRPAEY DNAAHQLVKRPEQF DAMLQQVLLRPAEY
316 325 QVGGIGIAPGAN LIGGLGFAPSAN QVGGIGIAPGAN
DAMAMHLVRSPARF DAMAMHLVKNPARF DNATMQLIKDPSQF DNATMQLIKDPSQF DNAAMQLIYAPNQF DNAAMQLIVNPKQF
LPGSLGLLPSAS LPGSLGLLPSAS ITGSMGMLPSAS ITGSMGMLPSAS
DNSVLKVVTNPSAY
*
*
VFZATHGTAPKYAGKNKVNPGSVILS
338 395 TVTYDFERLM VLTGDVVGYD TVTYDFERLM
IPGSLGLLPSAS IPGSLGLLPSAS
VFEPVHGSAPDIAGKGIANPTAAILS VFZPVHGSAPDIAGKGIANPTAAILS LYZPAGGSAPDIAGKNIANPIAQILS LYEPAGGSAPDIAGKNIANPIAQILS LFZPVHGSAPDIAGKGMANPFAAILS LYEPSGGSAPDIAGKGVANPIAQVLS MYEPVHGSAPDIAGKSIANPIAMIAS LYEPCHGSAPDL.GKQKVNPIATILS LYEPCHGSAPDL.PKNKVNPIATILS LYEPCHGSAPDL.PANKVNPIATILS LVEPIHGSAPDIAGKGIVNPVGTILS
TPPPDLGGSA TPPPDLGGSA IRTGDLARGA VRTGDLARGA KRTRDLARSE KRTRDIEVGS IRTADIMADG ITTADIGGSS IRTGDLGGSN IRTGDLKGTN LYTRDLGGEA
SAGSLGLTPSAN
IFZAVHGSAPDIAGQKDANPTALLLS
NRTGDLAGTA
LTGSLGMLPSAS ITGSIGMLPSAS LTGSLGMLPSAS IPGSLGLLPSAS IPGSLGLLPSAS
DAGGMQLVRKPKQF DSAAMILIKQPSKM DSAAMILVKNPTHL DSAAMILIKYPTQL DSAAMLLVKSPRTL
*
334 344 351 357 LFEATHGTAPKYAGQDKVNPGSIILS IFEAVHGSAPKYAGKNVINPTAVLLS
NAD-dependent TDH P. putida DILCARFVLQPERF CAGTIGIAPSAN SVTPDMGGTL 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, Schizosaccharomyces pombe, and Salmonella typhimurium (7). TDH, tartrate dehydrogenase (9). P. putida, Pseudomonas putida.
alent in the (3-turn of IMDH. If sequence alignments suggest that Arg occupies this "site" in several IMDHs, the secondary structure of the (3-turn in IMDH shows that the side chain will point away from the nucleotide binding site. Sequence alignments 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 was detectable). However, the performance with NAD was reduced below that of the wild-type enzyme (K344D/Y345I/ V351A/Y391G/R395G had a Km of 1400 pLM, a kcat of 0.284 sec-1, and a performance kcat/Km of 0.0002 ,uM-1lsec-1). A second mutant (K344D/Y345I/V351A/Y391R/R395A) produced comparable results. Instead, Tyr-391 was replaced by hydrophilic Lys that, being shorter than the Arg found in many IMDHs, 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 might generate steric effects with adjacent residues. Hence, Ser, a polar residue suitable for replacements at the surface of a protein, was chosen to replace Arg-395. The introduction of Y391K and R395S to generate K344D/Y345I/V351A/ Y391K/R395S caused a dramatic decrease in performance with NADP, as expected from the loss of two hydrogen bonds to the 2'-phosphate and a modest increase in performance with NAD (Table 2). The preference for NAD over NADP was thus 100-fold. A shift in the loop formed by residues 316-322 alters the orientation of Leu-320 in IMDH, nudging the adenosine moiety of NAD toward Arg-344. This shift, generated by Pro-317, is stabilized by a hydrogen bond between Asp-392 and the hydroxyl of Ser-319 that precisely replaces by a bound water 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 Performance,
,uM
kcat,1
sec-
M-1 . sec-I1
17 7,300 6,400 11,300 32,200 2,800 5,800
80.5 6.3 18.0 2.0 0.39 3.34 4.70
4.7 0.00086 0.0028 0.00018 0.000012 0.0012 0.00081
Km, Enzyme E. coli IDH at 21°C abcdefgh KYVYRRCC (wild type) DI -----DIA ----DIAKS--DIAKSD-DIAKS-YDIAKS-YI
Saccharomyces cerevisiae Wild-type IDH at 24°C (16, 17) Wild-type IMDH at 30°C (18) Salmonella typhimurium Wild-type IMDH at 24°C (19) T. thermophilus Wild-type IMDH at 65°C (7)
kcat/Km,
Km,
NAD Performance
kcat,
kcat/Km,
Preference, NADP
performance
uM
sec- 1
.&M-1-sec-1
/NAD performance
4700.0 3300.0 850.0 290.0 924.0 108.0 99.0
3.22 2.59 9.39 6.00 9.74 11.4 16.20
0.00069 0.00078 0.011 0.021 0.011 0.106 0.164
6900 1.1 0.25 0.009 0.001 0.011 0.005
210 140
40 14.48
0.190 0.103
100
31.5
0.315
29.9 0.00243 40 13.6 0.34 0.007 12,300 All apparent standard errors are 106: performance with NADP was reduced 6000-fold, and performance with NAD increased 240-fold (Fig. 2). Of the seven successful substitutions introduced into IDH, only two were based on strict homology with the NAD-dependent enzymes (Table 1). Our final mutant displays a preference for NAD over NADP of 200-fold; has an NAD Michaelis constant of 100 ,tM, representing an improvement by a factor of 50, and a kcat of 16.2 sec-1, which represents an increase by a factor of 5 (Table 2). The results suggest that a hydrogen bond between the adenosine ribose of NAD and Asp-344, as seen in the x-ray structure of the IMDH binary complex, may have been successfully established. Note that the Michaelis constant of isocitrate remains unchanged at 10 ,uM, suggesting that these mutations have no effect on substrate binding. The kinetic characteristics of the final engineered enzyme compare favorably with natural NAD-dependent IDHs and
Proc. Natl. Acad. Sci. USA 92 (1995) IMDHs from various sources (Table 2). The fact that the Michaelis constant of the mutant enzyme is higher toward NAD than that of the wild-type enzyme toward NADP appears typical of natural IDHs: the Michaelis constants of NADdependent enzymes range from 150 ,uM to 800 j,M, whereas those of the NADP-dependent enzymes range from 2 ,M to 20 ,uM (22). Perhaps the strong electrostatic interactions with the 2'-phosphate of NADP, evident in the crystal structure of E. coli IDH with NADP (Fig. 1B), are responsible for this difference. The reason why the maximum rate of catalysis displayed by wild-type IDH utilizing NADP is 5-fold higher than with the engineered mutant (Table 2) is unclear. Conformational changes induced by NADP binding in IDH are restricted to side-chain movements in the immediate vicinity of the coenzyme binding pocket (5). The structure of a pseudoMichaelis ternary complex with NADP and isocitrate has been determined (23), but again, no obvious changes in the active site can be ascribed to changes induced in the coenzyme binding pocket, which lies some 14 A away. Hence, our final mutant is probably as efficient an NAD-dependent IDH as is currently possible to design. We thank Eric Walters, Jim Hurley, and Bob Kemp for their thoughtful suggestions. This work was supported by Public Health
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