Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2005, p. 3285–3293 Vol. 71, No. 60099-2240/05/$08.00�0 doi:10.1128/AEM.71.6.3285–3293.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cooperative Effect of Two Surface Amino Acid Mutations (Q252L andE170K) in Glucose Dehydrogenase from Bacillus megaterium IWG3

on Stabilization of Its Oligomeric StateSang-Ho Baik,1,3* Fabrice Michel,2 Nushin Aghajari,2 Richard Haser,2 and Shigeaki Harayama1,3

Marine Biotechnology Institute, 3-75-1 Heita Kamaishi, Iwate 026-0001, Japan1; Institut de Biologie et Chimie des Proteines,UMR5086-CNRS, Laboratoire de BioCristallographie, IFR128 BioSciences Lyon-Gerland, 7 Passage du Vercors, 69367

Lyon cedex 07, France2; and Biotechnology Development Center, National Institute of Technology and Evaluation,2-5-8 Kazusa-Kamatari, Kisarazu-shi, Chiba 292-0818, Japan3

Received 11 August 2004/Accepted 21 December 2004

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252Lsubstitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381–6385, 1989) is stable atpH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stabilityat an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O.Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329–335, 2003). This mutant had two amino acidsubstitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring thesubstitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring twosubstitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K,were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomericstate of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution(Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L andE170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystalstructure of the E170K/Q252L mutant, determined at 2.0-A resolution, showed that residues 170 and 252 arelocated in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in thewild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutionsincrease the subunit-subunit interactions by stabilizing the hydrophobic cavity.

Glucose dehydrogenase (GlcDH) (E.C. 1.1.1.47) from Ba-cillus megaterium IWG3 is an oligomeric tetramer composed offour identical subunits with a molecular mass of 28 kDa, and itis a member of the short-chain alcohol dehydrogenase family(9, 8, 14). Wild-type GlcDH is inactivated at pH values above9 in the absence of a high concentration (2 M) of NaCl. At suchpH values, the quaternary structure of GlcDH is significantlyperturbed, as the tetrameric enzyme dissociates into inactivemonomers due to weak subunit-subunit interactions. When thepH is brought to neutral, the enzyme is reversibly renatured byreconstruction of the oligomeric tetramer (16, 17, 22). Al-though the dissociation and association of the tetrameric oli-gomer of wild-type GlcDH accompanying changes in pH or theurea concentration have been well characterized (18), the de-tailed mechanisms of the subunit-subunit interactions of GlcDHmust still be elucidated.

Several amino acid residues that affect the stability of thisenzyme have been identified by isolating GlcDH mutants ex-hibiting improved thermostability (1, 15, 18, 19). One of themutations giving rise to improved thermostability is Q252L.However, the Q252L mutant showed dramatically decreasedstability at alkaline pHs in the absence of a high concentrationof NaCl (15, 19). Recently, we isolated a GlcDH mutant withtwo substitutions, E170K and Q252L. The stability of the

Q252L/E170K enzyme at alkaline pHs was high even in theabsence of NaCl.

In this study, we examined the effect of the E170K andQ252L mutations on the secondary and quaternary structuresof GlcDH. Moreover, the crystal structure of the Q252L/E170K enzyme was determined and compared with the struc-ture of wild-type GlcDH which has been determined previ-ously (30). The results obtained in this study indicated that thestability of the Q252L/E170K GlcDH is driven by stabilizationof the quaternary structure and that a hydrophobic cavity isformed at the subunit-subunit interfaces and is stabilized bythe E170K and Q252L mutations.

MATERIALS AND METHODS

Site-specific mutagenesis. The L252Q substitution (wild-type allele) was in-troduced into the Q252L/E170K mutant gene by PCR-based site-specific mu-tagenesis to obtain the E170K mutant gene. The template was pTDN-46 carryingthe Q252L/E170K mutant gene (20), and the PCR amplification was carried outwith two primers, primers A (5�-ATGCAGCAAGTAAAGGCG3�) and B (5�-AAGTCGACTTATTCGCGTTCTGCTTGGAATGATGGGTACTGTGTCATACCG-3�). The residue of primer B that leads to the L252Q mutation is under-lined, while the SalI site in primer B is indicated by boldface. The PCR-amplifiedDNA fragment was restricted by the SalI restriction endonuclease (New EnglandBiolabs), and the resultant 322-bp fragment was isolated from an agarose gelwith a gel purification kit (QIAGEN). The isolated DNA fragment was ligated toSalI-restricted pTDN-46 to produce the gene encoding a mutant GlcDH carryingE170K. The mutation was confirmed by dye termination cycle sequencing usinga 377 DNA sequencer (Applied Biosystems).

Purification of GlcDHs. The GlcDHs carrying the Q252L, E170K, and Q252L/E170K mutations were overexpressed in Escherichia coli JM109 and purified byanion-exchange chromatography using a high-pressure liquid chromatographysystem (Tosoh). The active fraction was heated in 10 mM phosphate buffer (pH

* Corresponding author. Mailing address: Biotechnology Develop-ment Center, National Institute of Technology and Evaluation, 2-5-8Kazusa-Kamatari, Kisarazu-shi, Chiba 292-0818, Japan. Phone: 81-438-20-5908. Fax: 81-438-20-5766. E-mail: [email protected].

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6.5) at 60°C to remove heat-labile proteins of E. coli, and soluble proteins werepurified further by gel filtration as described previously (1, 19). For purificationof the Q252L and E170K enzymes, heat treatment at 60°C was carried out in 10mM phosphate buffer (pH 6.5) containing 2 M NaCl to prevent inactivation ofthe enzymes at this temperature. The soluble proteins after the heat treatmentwere dialyzed overnight at 4°C against 10 mM phosphate buffer (pH 6.5) toremove the excess NaCl before gel filtration. The purity of the purified enzymeswas checked by sodium dodecyl sulfate–12.5% polyacrylamide gel electrophore-sis at a constant current of 15 mA, and the enzymes were visualized by Coomassieblue or silver staining (11).

Enzyme assay. Under the standard assay conditions, the GlcDH activity wasdetermined spectrophotometrically at 25°C by measuring the rate of NADHformation at 340 nm in 100 mM Tris-HCl (pH 8.0) containing 0.1 M D-glucoseand 0.5 mM NAD� as described previously (1, 30). One unit of GlcDH wasdefined as the amount of the enzyme that produced 1 �mol of NADH per minunder the standard assay conditions. The protein concentration was determinedby the bicinchoninic acid method (28) with bovine serum albumin as a standard.The analyses of kinetic constants and substrate specificity were carried out asdescribed previously (1).

Enzyme inactivation at alkaline pH. Enzyme inactivation was determined at25°C by incubating the enzyme (10 �g ml�1) in 10 mM phosphate buffers at pHsranging from 8 to 10.5 and measuring the residual activity in 100 mM Tris-HCl(pH 8.0).

CD analysis. Circular dichroism (CD) spectra were obtained with a J-725 spec-tropolarimeter (Jasco) with a cell having a 1-cm path length at 25°C. The proteinsamples were continuously stirred to keep the temperature uniform. To monitor thealkali-induced denaturation of the secondary structures of GlcDHs, changes in theCD spectra around 222 nm were recorded in 10 mM phosphate buffer (pH 6.5)before and after incubation of the enzymes at alkaline pHs as described above.

Quaternary structure analysis by gel filtration chromatography. Purified en-zymes (300 �g) were incubated in 10 mM phosphate buffer (pH 6.5) at pH valuesbetween 8 and 10.5 for 1 h at 25°C and loaded onto a Superdex R 200HR 10/30column (Amersham Biosciences) which had been equilibrated with 10 mMpotassium phosphate buffer at pH values between 8 and 10.5 and fitted to aTosoh high-pressure liquid chromatography system (Tosoh). The proteins wereeluted at a flow rate of 0.4 ml/min. For equilibration to a specific pH, the columnwas washed with buffer at the pH overnight at a flow rate of 0.4 ml/min. Theprotein elution profile was monitored at 280 nm at room temperature.

Crystallization of the Q252L/E170K mutant. Crystallization was performedusing the hanging-drop vapor diffusion method with the Q252L/E170K enzyme (10mg/ml) in the presence of NAD� (4 mM) in 50 mM sodium phosphate buffer (pH6.3) as described for the native enzyme by Yamamoto et al. (30). Crystals weregrown from 0.1 M morpholineethanesulfonic acid (MES) (pH 6.5) and 30% (wt/vol)polyethylene glycol 6000 at 18°C and appeared within 3 to 4 weeks.

Data collection and model refinement. Crystals were flash frozen at 100 K ina nitrogen stream before data collection using 50% ethylene glycol as a cryo-protectant. Diffraction data were collected at 2.0-A resolution with a MarCCDdetector at European Synchrotron Radiation Facility beamline BM30A (� �0.98 A). Diffracted intensities were integrated with the program MOSFLM (13),as implemented in the CCP4 software package and scaled with SCALA (4).Refinement of the model was done with the simulated annealing protocol asimplemented in the software package CNS (3) using all reflections between 15and 2.0 A and then alternating cycles of positional refinement and manualbuilding using TURBO-FRODO (23). Water molecules present at similarpositions in the Q252L/E170K mutant and wild-type structures have the samenumbering. Free and conventional R factors were monitored (2) to avoidoverrefinement. Model qualities were examined with PROCHECK (12) andWHATCHECK (6).

Protein structure accession number. Atomic coordinates and structure factorshave been deposited in the Protein Data Bank (PDB) under accession code 1RWB.

RESULTS

Enzyme inactivation at alkaline pHs and changes in thesecondary structures of GlcDHs. In this study, we constructeda gene encoding GlcDH possessing the E170K mutation. Themutant enzyme was expressed in E. coli and purified. Thekinetic constant of the E170K enzyme was determined and isshown in Table 1 together with those of the Q252L and Q252L/E170K enzymes. We investigated the stabilities of these threemutant enzymes at pH values ranging from 8 to 10.5. Theenzymes were incubated in 10 mM phosphate buffer at eachpH for 1 h, and the residual activity was measured under thestandard assay conditions. As reported previously, the Q252Lenzyme was inactivated at pH values above 9 (19). The newly

FIG. 1. Stability of glucose dehydrogenase at different pHs. Theactivities of the wild-type enzyme (A) and three mutant enzymes, theQ252L (B), E170K (C), and Q252L/E170K (D) enzymes, were mea-sured after incubation of the enzymes for 1 h at various pHs in theabsence (open bars) or presence (dotted bars) of 2 M NaCl. The errorbars indicate standard errors of the means (n � 4).

TABLE 1. Apparent kinetic constants of glucose dehydrogenases harboring E170K, Q252L, and E170K/Q252L substitutionsa

Enzyme Apparent Km (Glu)(mM)

Apparent Km (NAD�)(mM)

Apparent Km (NADP�)(mM)

Apparent kcat (Glu)(s�1)

Apparent kcat (Glu)/apparent Km(Glu) (mM�1 s�1)

Wild type 14 0.17 0.033 395 28Q252L 8 0.15 0.043 317 40E170K 7.5 0.22 0.051 352 47Q252L/E170K 8.5 0.33 0.049 334 39

a Apparent kinetic constants were determined as described previously (1). The enzyme activity was measured with a Beckman DU-7500 spectrophotometer equippedwith a thermostatic circulating water bath. The D-glucose concentrations for Km determinations were varied from 0.1 mM to 20 mM in 50 mM Tris-HCl buffer (pH8.0) containing 0.5 mM NAD�, while the Km values for NAD� and NADP� were determined with 0.15 to 2.0 mM NAD� and 0.05 to 2 mM NADP in 50 mM Tris-HClbuffer (pH 8.0) containing 0.1 M glucose.

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isolated mutant enzyme carrying E170K was shown to be rel-atively resistant to alkaline pHs, as shown in Fig. 1. Approxi-mately 26% of the initial enzyme activity remained even afterincubation at pH 10 or 10.5. The Q252L/E170K enzyme wasmore stable than the E170K enzyme and retained more than95% of the initial enzyme activity after treatment at pH valuesbetween 8 and 10.5. The results indicated that there was asynergetic effect of the E170K and Q252L mutations thatmaintained the structural integrity of GlcDH at alkaline pHs.On the other hand, the stabilities of the Q252L and E170Kenzymes at alkaline pHs were significantly increased when 2 MNaCl was added, and they were indistinguishable from thestability of the Q252L/E170K enzyme.

Although the results shown in Fig. 1 clearly demonstrat-ed the difference in the stabilities of the Q252L, E170K, andQ252L/E170K enzymes, it should be mentioned that the assaysprobably overestimated the stabilities of these enzymes at al-kaline pHs. It is known that wild-type GlcDH shows reversibledissociation-association between inactive monomers and activetetramers when the pH is shifted between 9 and 7 (16, 17).

Thus, a fraction of an inactivated enzyme might be reactivatedduring enzyme assays conducted at pH 8.0.

The enzymes were analyzed by CD spectroscopy. A localnegative maximum in ellipticity was observed around 226 nm inthe spectra of all enzymes before denaturation. As shown inFig. 2, the alpha-helix content of each enzyme (monitored at222 nm) did not change significantly at pH 9.5, at which theQ252L enzyme was completely inactivated and the E170K en-zyme was partially inactivated, indicating that the inactivationof this enzyme at pH 9.5 was not due to the loss of secondarystructures. At pH 10.5, the alpha-helix contents of the mutantQ252L and E170K enzymes slightly decreased, as shown in Fig.2, suggesting that prolonged incubation of these enzymes at pH10.5 resulted in partial unfolding of the secondary structures.In contrast, the secondary structures of the double-mutantQ252L/E170K enzyme were not affected even at pH 10.5, il-lustrating that the Q252L/E170K double mutant was more resis-tant to alkaline pHs than the E170K and Q252L single mutants.

Gel filtration chromatography. Since inactivation of thewild-type GlcDH at an alkaline pH was proven to be stronglycorrelated with destruction of the quaternary structure (tet-ramer) of the enzyme (16, 17, 22), gel filtration chromatogra-phy was carried out to analyze the enzyme carrying either theQ252L, E170K, or Q252L/E170K mutation(s) before and afteralkali-induced inactivation. The native forms of these threeenzymes at neutral pH were eluted as a single peak around 110

FIG. 2. Far-UV circular dichroism spectra of the Q252L, E170K,and Q252L/E170K enzymes. Far-UV circular dichroism spectra wereobtained with the enzymes incubated at pH 8.0 (thick lines), pH 9.5(thin lines), and pH 10.5 (dashed lines) for 1 h. Protein concentrationswere adjusted to 0.1 mg/ml in the same buffers (10 mM), and thespectra obtained were processed using the smoothing function in-stalled in a standard Jasco analysis program. (A) Q252L enzyme; (B)E170K enzyme; (C) Q252L/E170K enzyme.

FIG. 3. Gel filtration chromatography analyses of glucose dehydro-genase mutants after incubation at various pHs. (A) Q252L/E170Kenzyme; (B) E170K enzyme; (C) Q252L enzyme. The standard molec-ular mass markers used to establish the calibration curve were bovinethyroglobulin (670 kDa), glutamate dehydrogenase (290 kDa), lactatedehydrogenase (142 kDa), enolase (67 kDa), chicken ovalubumin (44kDa), myokinase (32 kDa), horse myoglobin (17 kDa), vitamin B12(13.5 kDa), and cytochrome c (12.4 kDa; Amersham Biosciences).

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kDa, a size corresponding to a tetramer (Fig. 3). The Q252L/E170K enzyme, which was stable at alkaline pHs, showed ab-solutely identical chromatographic profiles before and afterthe alkali treatment, as shown in Fig. 3. On the other hand, twopeaks at 110 and 60 kDa, corresponding to the tetrameric anddimeric oligomers, respectively, were detected with the Q252Lenzyme after treatment at pH 8.5 in 10 mM potassium phos-phate buffer, conditions under which only 50% of the enzymeactivity was retained. Moreover, when the pH was shifted to9.5, the tetramer peak of the Q252L enzyme disappeared com-

pletely, and there was a single peak at 60 kDa corresponding tothe dimeric state (Fig. 3). The intensity of the dimer peak aftertreatment at pH 10.5 was significantly lower than that at pH9.5, indicating partial unfolding of the secondary structures oraggregation. In the case of the E170K enzyme, no change inthe elution pattern was observed after treatment at pH 8.5. AtpH 9.5, two peaks of the E170K enzyme corresponding to thetetrameric and dimeric oligomers appeared, while at pH 10.5,a single peak corresponding to a dimer was observed. No traceof monomers was present under these conditions.

FIG. 4. (A) Crystal structure of the Q252L/E170K enzyme with a close-up of the region harboring Leu252 and Lys170. Monomers B, C, andD are grey, while monomer A is different colors. Leu252, Lys170, and NAD� are represented by ball and stick models and are color coded by atomtype (yellow, carbon; red, oxygen; blue, nitrogen). Leu252 and Lys170 on monomer A are indicated by larger spheres. The P-axis interface isbetween molecules A and D, whereas the Q-axis interface is between molecules A and B. Helices in monomer A are different colors, ranging fromblue at the N terminus and then green followed by yellow and finally red at the C terminus. (B) Superimposition of the Q252L/E170K (black lines)and wild-type (grey lines) glucose dehydrogenases. Direct interactions between amino acids are indicated by thick dashed lines, and interactionsmediated by water molecules are indicated by thin dashed lines. The figures were produced with the program MOLSCRIPT (10).



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Three-dimensional structure determination. Crystals of theQ252L/E170K enzyme belonged to monoclinic space groupC2, and their unit cell parameters were quite similar to thoseof the wild-type GlcDH structure. Diffraction data were col-lected to 2.0 A, and the statistics are summarized in Table 2.Since the crystals of the Q252L/E170K enzyme were isomor-phous with those of wild-type GlcDH, the three-dimensionalstructure of the mutant was determined by using the phases ofthe wild-type enzyme structure as solved at 1.7-A resolution(30), in which NAD� as well as water molecules had beenremoved in order to avoid bias. The refined model (Rcryst �22.4%, Rfree � 26.6%) contained 1,044 amino acid residues,549 water molecules, and four NAD� molecules. The details ofthe refinement results are given in Table 2.

As expected, the Q252K/E170K enzyme, like the wild-typeGlcDH, consisted of four identical subunits, each displaying aseven-stranded parallel �-sheet flanked by two arrays of three�-helices (Fig. 4A). The sites of substitutions within the Q252Land E170K enzymes were surrounded by several hydrophobicamino acid residues, which formed a hydrophobic cavity-likeinterface of oligomeric structures, as shown in Fig. 4B. Thehydrophobic cavity around residue 252 of monomer A [252(A)] is lined up by residues Y253 (A), P214 (A), M215 (A),P254 (A), F256 (A), I150 (C), P151 (C), W152 (C), A173 (D),and L174 (D). In both wild-type and mutant enzymes, there is

an interaction between Y253 (A) and T251 (C) at one edgeof the hydrophobic cavity. In the wild-type enzyme, Q252(A) interacts with G241 (D) to make intermolecular con-tacts across the P axis, as defined in Yamamoto et al. (30). Thisinteraction was not observed in the Q252L/E170K structure.Instead, in the mutant enzyme, G241 (D) forms intramolecularcontacts with K170 (D) bordering another edge of the hydro-phobic cavity. Moreover, in the latter mutant enzyme, twowater molecules (Wat269 and Wat320 according to the nomen-clature of the wild-type structure) observed around residue 252in the wild-type structure were lacking (Fig. 4B).

DISCUSSION

In a search for a more stable GlcDH for determination ofthe blood glucose level in clinical diagnosis and for coenzymeregeneration in various biotechnological applications, we pre-viously isolated a thermostable GlcDH double mutant with theQ252L and E170K substitutions. Since the mutated amino acidresidues are located at the monomer-monomer or dimer-dimerinterfaces, as shown in Fig. 4, we expected these mutations tobe involved in stabilization of the oligomeric structure of theenzyme (30). In the present study, we showed by using gelfiltration chromatography and circular dichroism analyses thatinactivation of the enzyme carrying either the E170K or Q252L

FIG. 4—Continued.



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mutation occurred with dissociation of the oligomeric structurefrom tetramer to dimers without a significant change in the CDspectra. In contrast to an enzyme carrying a single mutation(either E170K or Q252L), the enzyme carrying both mutationsin a single molecule was stable in terms of activity, as well asquaternary structure, under all conditions examined. At pH10.5 in the absence of 2 M NaCl, more than 90% of the E170Kenzyme was dissociated into dimers (Fig. 3), while the residualactivity of the E170K enzyme incubated under the same con-ditions was 26% (Fig. 1). We thought that the inconsistencybetween the tetramer content and the enzyme activity of theE170K enzyme might have been due to reactivation of inacti-vated enzymes during the enzyme assay at pH 8.0. To test thishypothesis, we carried out an experiment in which the E170Kenzyme was incubated at either pH 8.0 or 10.5 for 1 h, and theresidual activity was assayed at pH 10.5. The E170K enzymeincubated at pH 8.0 showed activity, and the activity decreasedgradually at pH 10.5, indicating that enzyme inactivation tookplace during the measurement of activity at pH 10.5. TheE170K enzyme incubated at pH 10.5, in contrast, showed verylow activity at pH 10.5. Although it was difficult to measureaccurately the enzyme activity at pH 10.5, the residual activityof the E170K enzyme incubated at pH 10.5 was estimated to beless than 10% of the activity of the enzyme incubated at pH 8.0(unpublished data),

Thus, a striking difference in stability between the E170K,Q252L, and Q252L/E170K mutants was observed at alkalinepHs in the absence of a high concentration of NaCl. In theseconditions, the stabilities of the GlcDHs were as follows:Q252L/E170K enzyme E170K enzyme Q252L enzyme wild-type enzyme. Although the stability of the Q252L enzymewas almost identical to that of the wild-type enzyme at alkalinepHs and in the absence of NaCl, its thermostability was im-proved at neutral pHs (15, 19). Since residue 252 is located atthe interface involved in the association between subunits Aand D (Fig. 4B), increased stability of the Q252L enzyme athigh temperatures was expected to be related to the reinforce-ment of the subunit association around the P axis due to theQ252L mutation. On the other hand, the E170K mutation,located near the interface between subunits A and B (Q axis),improved the stability at alkaline pHs, suggesting that theQ-axis association is stabilized by the E170K mutation. Inorder to understand the details of the stabilization mechanisms

induced by these two mutations, we determined the crystalstructure of the E170K/Q252L enzyme at 2.0-A resolution(Fig. 4A) and compared it to the wild-type three-dimensionalstructure.

On the basis of the crystal structure, we propose that L252 ofthe mutant enzyme stabilizes the tetramer structure by chang-ing the environment in the vicinity of Y253. The latter residueforms intermolecular contacts, especially with T251 [namely,Y253 (A)-T251 (C)] in both the wild-type and mutant enzymes.Y253 is in a conformation with dihedral angles (�, �) charac-teristic of a left-handed helix, meaning that the carbonylgroups of both residues 252 and 253 point in the same direc-tion. The side chain carbonyl carbon of Q252 in the wild-typeenzyme is in a hydrophobic environment; thus, the side chainof L252 in the mutant enzyme may strengthen the hydrophobicnature of this region. One edge of this region is closed by the

TABLE 2. Data collection and refinement statistics

Data set Data

Space group ..............................................C2Cell parameters ........................................a � 118.7 A, b � 65.4 A, c �

118.0 A, � � 92.8°Beamline at ESRFa .................................BM30AResolution (A) .........................................30.0–2.0Completeness (%)b..................................99.4 (99.4)Total no. of observations ........................193,166No. of unique reflections ........................61,998Redundancyb ............................................3.1 (3.1)Rsym (%)b,c ................................................5.7 (11.9)I/ b .............................................................7.5 (5.9)Refinement

Resolution (A) .....................................15.0–2.0Total no. of protein atoms .................7,884No. of water molecules .......................549

R.m.s.d. from ideal geometryBond length (A)...................................0.006Bond angle (°) ......................................1.2

R factor (%) (reflections)d .....................22.4 [60,614]Rfree (%) (reflections)e ............................26.7 [6,090]

a ESRF, European Synchrotron Radiation Facility.b Data for the highest-resolution shell are given in parentheses.c Rsym � ��I � � I ��I, where I is the intensity of the individual reflections and

� I � is the mean intensity over symmetrically equivalent reflections.d R � ��Fobs� � �Fcalc�I��Fobs�, where Fobs and Fcalc are the observed and

calculated structure factor amplitudes, respectively.e Rfree is the same calculation including only the randomly chosen 10% of

reflections not used for refinement.

FIG. 5. Protein sequence alignment based on the three-dimensional structures of enzymes belonging to the short-chain alcohol dehydrogenasefamily. The secondary structure of the B. megaterium glucose dehydrogenase (PDB accession code 1GCO) is shown above the sequence alignment.Strongly conserved residues are enclosed in boxes, in which white letters with a red background indicate absolutely conserved residues and residueswith similar biochemical properties are indicated by red letters. Residues equivalent to residue 170 or 252 in the 1GCO sequence are indicatedby stars. Positively charged residues at positions equivalent to E170 in the 1GCO structure are indicated by a yellow background, while hydrophobicresidues at positions equivalent to Q252 are indicated by a green background. The triad of catalytically important residues (S, Y, and K) in theshort-chain alcohol dehydrogenase family are indicated by triangles, and the N-terminal coenzyme-binding motif (G-X3-G-X-G) is indicated bysolid circles. The enzymes corresponding to PDB codes are as follows: 1GEG, Klebsiella pneumoniae acetoin reductase; 1EDO, Brassica napus�-keto acyl carrier protein reductase; 1DOH, Magnaporthe grisea trihydroxynaphthalene reductase; 1GCO, B. megaterium glucose dehydrogenase;1HXH, Comamonas testosteroni 3�/17�-hydroxysteroid dehydrogenase; 1HDC, Streptomyces exfoliatus 3�,20�-hydroxysteroid dehydrogenase;1AHH, E. coli 7�-hydroxysteroid dehydrogenase; 1AE1, Datura stramonium tropinone reductase I; 2AE1, Datura stramonium tropinone reductaseII; 1E3S, Rattus norvegicus short-chain 3-hydroxyacyl-coenzyme A dehydrogenase; 1CYD, Mus musculus carbonyl reductase; 1H5Q, Agaricus bisporusNADP-dependent mannitol dehydrogenase; 1BDB, Pseudomonas sp. cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase; 1FJH, Comamonas testosteroni3�-hydroxysteroid dehydrogenase/carbonyl reductase; 1A27, Homo sapiens 17-�-hydroxysteroid dehydrogenase; 1A4U, Drosophila alcohol dehydroge-nase; 1NAS, Mus musculus sepiapterin reductase; and 1DHR, Rattus norvegicus dihydropteridine reductase. The relative accessibility of eachresidue (blue, accessible; cyan, intermediate; white, buried) and the hydropathic character (pink, hydrophobic; grey, intermediate; cyan, hydrophilic) ofthe 1GCO sequence are shown below each block. The sequence alignment was generated by CLUSTALW (26) and displayed with ESPript (5).



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interaction between Y253 (A) and T251 (C) in the wild-typeenzyme, while another H-bond interaction bordering the cavityis found in the mutant enzyme between K170 (D) and G241 (D).

Sequence alignments of enzymes that belong to the short-chain alcohol dehydrogenase family and whose three-dimen-sional structures are known show that only 2 of 18 sequencescontain the glutamine mentioned above at position 252, where-as 12 sequences contain a hydrophobic residue at this position(Fig. 5). Therefore, it seems that hydrophobic residues are pref-erable at this position in order to stabilize the oligomeric struc-ture.

In our previous study (1), we assumed that anion-anionrepulsion between subunits is a main reason for the instabilityof the wild-type and Q252L enzymes under alkaline conditionsand in the absence of a high concentration of NaCl. Since thestability of GlcDH under alkaline conditions was significantlyimproved by the E170K mutation, residue 170 seems to becrucial for the stability at alkaline pHs. This residue is locatedon the �E helix, which belongs to the Q-axis-related interface.In the wild-type enzyme, E170 interacts with K149 across the Qaxis between the A and B subunits and between the C and Dsubunits via water-mediated hydrogen bonds to Wat41 andWat56. In the structure of the E170K/Q252L mutant, the watermolecules mentioned above are displaced, and the water-me-diated interactions between two subunits across the Q axis areabsent (Fig. 4B). Moreover, the side chain of K170 (D subunit)forms direct hydrogen bonds with the carbonyl oxygen of G241(D subunit), resulting in a new intramolecular interaction.Thus, the E170K mutation probably increases the hydropho-bicity of this region in relation to the longer side chain of alysine compared to a glutamic side chain. Therefore, the Lysresidue may help prevent access of water molecules to thehydrophobic core due to steric hindrance and to direct hydro-gen bonding to the carbonyl oxygen of G241.

The profile of the pH-dependent inactivation of GlcDHshown in Fig. 1 indicated that an ionizable species with a pKa

of 8 or more is crucial for the subunit-subunit interaction. Weassumed that E170 has an anomalously high pKa due to ahydrophobic environment that stabilizes the protonated formof this residue, as has been shown for other proteins (21, 27).We further assumed that the negative charge of E170 contrib-utes to disruption of the dimer-dimer association. This hypoth-esis explains why K170, under any conditions, renders GlcDHresistant to alkaline pHs. It also explains the salt-dependentstability of the wild-type and Q252L enzymes under alkalineconditions, suggesting that in the presence of a high salt con-centration, coulombic interactions are essentially screened out.

Sequence comparison of enzymes belonging to the short-chain alcohol dehydrogenase family (Fig. 5) showed that themajority of the amino acid residues at position 170 were basic(R or K) and that the E residue was found only in GlcDH(PDB accession code 1GCO) and in 17-�-hydroxylsteroid de-hydrogenase (PDB accession code 1A27).

In conclusion, the stabilization of the quaternary structure ofGlcDH by the E170K/Q252L mutations is interpreted to be theresult of the cooperative effects of these two mutations. Resi-due 252 is in a relatively unstable hydrophobic cavity sur-rounded by each of the monomers. This cavity appears to beeasily accessible by water molecules, and their presence in sucha hydrophobic core is certainly not thermodynamically favor-

able for stabilization of the surrounding region. As this hydro-phobic region interlinks the four monomers, its instabilitywould lead to instability of the active enzyme. On the otherhand, the Q252L mutation may contribute to stabilization ofthe hydrophobic core in two ways: by removing a carbonylgroup which is a potential hydrogen bond acceptor candidateand by displacing two water molecules from this region. TheE170K mutation may also help to stabilize the enzyme struc-ture by increasing the hydrophobicity around the Q-axis inter-face with the displacement of two water molecules in the vi-cinity of K170 and by preventing the development of a negativecharge at residue 170 under alkaline conditions.

Since many enzymes consist of oligomeric structures formaintaining their activities, it is important to elucidate thefactors involved in stabilization of their oligomeric states. Un-fortunately, our knowledge concerning these aspects is still farfrom complete. Generally, the oligomeric structure of proteinsis formed by various subunit-subunit interactions, includingdisulfide bonding, hydrogen bonding, and hydrophobic andelectrostatic interactions. To understand how to increase thestability of these oligomeric organizations, conditions whichincrease or decrease the stability of an oligomeric structurehave been investigated (7, 24, 25, 29). In this report, we pro-pose an efficient stabilization mechanism for GlcDH whichinvolves the shielding of a hydrophobic core by preventing itsinteractions with water molecules, a strategy which may beuseful for engineering other proteins.

ACKNOWLEDGMENTS

We thank Hideko Yoshida for technical assistance.This work was supported by the New Energy and Industrial Tech-

nology Development Organization (NEDO).

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Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase...

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