Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant

BBRC Biochemical and Biophysical Research Communications 347 (2006) 502–508 www.elsevier.com/locate/ybbrc

Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant q Takeo Tomita a, Shinya Fushinobu b, Tomohisa Kuzuyama a, Makoto Nishiyama a

a,*

Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

b

Received 8 June 2006 Available online 30 June 2006

Abstract To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase ˚ resolution of EX7 complexed with NADPH and NADH, mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2 0 -phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2 0 -phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.  2006 Elsevier Inc. All rights reserved. Keywords: Crystal structure; Malate dehydrogenase; Coenzyme specificity; Thermostable enzyme

Malate dehydrogenases (MDHs, EC 1.1.1.37) that catalyze the reversible conversion between malate and oxaloacetate are divided into two groups depending on the preference for the coenzyme. One is NAD(H)-dependent enzyme found in bacteria and non-plant organisms and the other is NADP(H)-dependent MDH (chMDH) found in plant chloroplasts [1,2]. In most cases, bacterial MDH is similar to mitochondrial MDH in amino acid sequence [3,4]. An MDH (tMDH) from an extremely thermophilic bacterium Thermus flavus AT-62 is an enzyme with the preference for NAD(H) [4–6]. Despite the NAD(H) preference, tMDH is more similar to chMDH than other bacterial and mitochondrial MDHs in amino acid sequence. The

q

Abbreviations: MDH, malate dehydrogenase; chMDH, chloroplast malate dehydrogenase; NHDH, nicotinamide hypoxanthine dinucleotide; NHDPH, nicotinamide hypoxanthine dinucleotide phosphate; PEG, polyethylene glycol; r.m.s.d., root mean square deviation; tMDH, malate dehydrogenase from Thermus flavus. * Corresponding author. Fax: +81 3 5841 8030. E-mail address: [email protected] (M. Nishiyama). 0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.131

N-terminal domain of MDH including tMDH has a Rossmann dinucleotide-binding fold. By replacing a loop region between b-strand B and a-helix C in the Rossmann fold, tMDH was successfully altered from an NAD(H)-dependent enzyme to an NADP(H)-dependent enzyme, named EX7 [7]. Alteration of coenzyme specificity of many kinds of NAD(P)H-dependent enzymes has been attempted later, most successfully for xylitol dehydrogenase from Pichia stipitis [8] and alcohol dehydrogenase from Rana perezi [9]. However, the structural basis for the alteration of the coenzyme specificity of NAD(P)H-dependent enzymes has been shown in only few cases: the alteration of coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenase from NADH toward NADPH [10] and 2,5-diketo-Dgluconic acid dehydrogenase from NADPH toward bi-dependency [11]. To elucidate the structural basis for the coenzyme specificity of these enzymes in detail, determination of their crystal structures in both NADH- and NADPH-bound forms is obviously required. However, a structure of the enzyme complexed with a non-preferable

T. Tomita et al. / Biochemical and Biophysical Research Communications 347 (2006) 502–508

503

coenzyme has never been reported. Recently, we determined the crystal structure of NADH-dependent tMDH in an NADPH-bound form. This binary complex, however, unexpectedly binds the coenzyme in the opposite orientation, where adenine occupies the position near the catalytic center and nicotinamide is positioned at the adenine binding site of the tMDH-NADH complex [12]. Therefore, it would be difficult to elucidate the structural basis for the coenzyme specificity of wild-type MDH. In this study, to reveal the reasons for the alteration of EX7 mutant coenzyme preference for NADPH over NADH, which would successively give a hint to elucidate the structural basis for the coenzyme specificity of wild-type tMDH, we deter˚ resolution of EX7mined the crystal structures at 2.0 A NADPH and EX7-NADH complexes. In this report, we show the crystal structures of both complexes and demonstrate in detail how EX7 discriminates between NADPH and NADH.

position 44. The mutated genes were expressed in Escherichia coli JM105 under the control of the lac promoter on pUC19 by induction with 1 mM isopropyl-b-D-thiogalactopyranoside. The mutants were purified as described previously [4,7]. Kinetic analysis. Steady-state kinetic analyses were carried out by measuring the decrease in absorbance in reaction mixtures containing 0.5 lg/ml of the purified wild-type enzyme or 1–1.5 lg/ml of the purified mutant enzymes in 100 mM sodium phosphate buffer (pH 7.0) at 30 C. In order to determine Km for NAD(P)H or nicotinamide hypoxanthine dinucleotide (phosphate) (NHD(P)H), concentrations of coenzymes were varied in the range of 2–200 lM by using a fixed concentration of oxaloacetate (50 lM) in the experiment for determining Km for NADH and NHDH, and a fixed concentration of oxaloacetate (25,000 lM) in the experiment for determining Km for NADPH and NHDPH. Data were analyzed with the HYPER program [19].

Materials and methods

The crystal structures of EX7-NADPH and EX7NADH complexes were determined by means of molecular replacement, using the structure of tMDH (PDB entry 1BMD) as a search model. Statistics for the data collection and refinement are shown in Table 1. The

Crystallization of EX7 mutant. The procedures for expression and purification of the EX7 mutant have previously been described [4]. The purified protein was concentrated up to 15 mg/ml in 20 mM Tris–HCl (pH 8.0) for crystallization. Crystals of EX7 were obtained by the vapor diffusion method with a reservoir solution containing 20–25% (w/v) polyethylene glycol (PEG) 4000, 1 mM dithiothreitol, and 100 mM Tris–HCl (pH 8.0–9.0) within 1 week at 20 C. Data collection and processing. Before data collection, the crystals were transferred to a reservoir solution finally supplemented with a cryoprotectant, 15% PEG 400 (w/v), and 1 mM NADPH or NADH by increasing the concentration of cryoprotectant to 15% by 5% in each step, with equilibration for 1 min between steps. Data from EX7-NADPH and EX7-NADH complex structures were collected with a CCD camera on the BL6A and NW12A station, respectively, at the Photon Factory, High Energy Accelerator Research Organization (KEK). All the data were collected at 95 K on a single crystal mounted in a cryoloop for each complex. Diffraction images were indexed, integrated, and scaled using HKL2000 [13]. The crystals of EX7-NADPH and EX7-NADH belong to space group P212121 with unit ˚ , b = 85.39 A ˚ , and c = 117.7 A ˚ and cell dimensions of a = 70.31 A ˚ , b = 84.94 A ˚ , and c = 116.94 A ˚ , respectively. The data collected a = 70.4 A ˚ were used for subsequent molecular replacement at the wavelength of 1.0 A and crystallographic refinement. Molecular replacement was performed with MOLREP [14] in the CCP4 program suite [15]. Model correction in the electron density map was performed with the XtalView program suite [16]. Refinement was performed with CNS 1.1 [17]. The final models of the EX7NADPH and EX7-NADH complexes contain the A- and B-chains of EX7 dimer and a part of an NAD(P)H molecule in each subunit and water molecules. Figures were prepared using XFIT in the XtalView program suite, Raster3D [18] and Pymol [http://pymol.sourceforge.net/]. The atomic coordinates have been deposited in the RCSB Protein Data Bank with Accession Nos. 1WZI and 1WZE. Site-directed mutagenesis and preparation of the mutants. Two additional mutants were constructed based on EX7 and EX3 as the templates by a QuickChange kit (Stratagene-Japan, Tokyo, Japan). Synthetic oligonucleotides, 5 0 -TTGGGGTCCGAGCAGAGCTTCCAGGCCCTG-3 0 and 5 0 -CAGGGCCTGGAAGCTCTGCTCGGACCCCAA-3 0 , were used as mutagenic primers for constructing EX6 which contains the amino acid sequence similar to that of EX7 but has the wild-type amino acid, Gln, in place of Arg at position 44. Synthetic oligonucleotides, 5 0 -CTTTTGGG ATCACCCCGATCCATGAAGGCC-3 0 and 5 0 -GGCCTTCATGGATC GGGGTGATCCCAAAAG-3 0 , were used as mutagenic primers for constructing EX4 which contains the amino acid sequence similar to that of EX3 but has the chloroplast-type amino acid, Arg, in place of Gln at

Results Overall structures of EX7-NADH and EX7-NADPH complexes

Table 1 Data collection and refinement statistics of EX7-NADPH and EX7NADH complexes

A. Data collection statistics Beamline Space group Cell dimensions ˚) a (A ˚) b (A ˚) c (A ˚) Resolutiona (A Total reflections Unique reflections Rsymb (%) I/r (I) Completeness B. Refinement statistics ˚) Resolution (A R-factor (%) Rfreec (%) No. protein atoms No. ligands No. water molecules R.m.s.d. from ideal values ˚) Bond length (A Bond angles (deg.) C. Average B-factor Protein Loop region Coenzyme a

EX7-NADPH

EX7-NADH

6A P212121

NW12 P212121

70.31 85.39 117.70 2.00 (2.00–2.11) 48,606 48,460 5.0 10.2 (4.9) 99.6 (100.0)

70.40 84.94 116.94 2.00 (2.00–2.07) 48,112 43,099 4.9 26.9 (12.5) 90.1 (84.8)

28.89–2.00 19.6 22.9 4976 2 624

48.16–2.00 19.3 23.5 4978 2 438

0.006 1.3

0.005 1.2

17.13 27.19 32.44

16.06 29.90 26.04

Highest resolution range for compiling statistics. PP P Rsym = jIi  ÆIæj/ ÆIæ. c Calculated using a test data set: 5% of total data selected randomly from observed reflections. b

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T. Tomita et al. / Biochemical and Biophysical Research Communications 347 (2006) 502–508

EX7-NADPH and EX7-NADH complexes are dimers consisting of identical subunits like tMDH-NADH. The overall structures of the complexes are similar to that of the tMDH-NADH complex [20] with root mean ˚ and 0.38 A ˚ for square deviations (r.m.s.ds) of 0.33 A the main chain atoms, respectively. Thus, the mutations have been accommodated with minimal overall structural changes. In the EX7 mutant, seven loop amino acids in positions 41–47, Glu-Ile-Pro-Gln-Ala-Met-Lys, are replaced by the corresponding loop residues, Gly-SerGlu-Arg-Ser-Phe-Gln, in the NADP(H)-dependent MDH from chloroplasts. In the crystal structures of the EX7-NADPH and EX7-NADH complexes, most residues of the loop region are clearly visible in the electron density map (Fig. 1) and the main chains of the loop regions have essentially the same structure with r.m.s.d. ˚ with each other. The main chain of the loop of 0.12 A region of the EX7-NADPH complex is also similar to those of the tMDH-NADH complex and the NADPH complexes of chMDH from Flaveria bidentis with ˚ and 0.35 A ˚ , respectively (Fig. 1B). r.m.s.d. of 0.37 A B-factors of the EX7-NADPH and the EX7-NADH ˚ 2 and 15.7 A ˚ 2, respectivecomplexes are in average 16.7 A ly, which are smaller than those of the tMDH-NADH ˚ 2) and the chMDH-NADPH complex complex (24.6 A 2 ˚ ). NADPH and NADH of the EX7 complexes (22.4 A are superimposable on NADH of the wild-type tMDH ˚ and 0.79 A ˚ , respectively, with an r.m.s.d. of 0.54 A although some portions of the coenzymes are invisible in the electron density maps as will be described below. A

Interaction of the loop region residues with NAD(P)H In the tMDH-NADH complex, Glu41 cO2 (OE1) and cO3 (OE2) hydrogen-bond to O2 0 and O3 0 atoms of the adenine ribose moiety of NADH [20] (Fig. 2C). Thus, the carboxyl group of Glu41 prevents the 2 0 -phosphate group of NADPH from binding due to electrostatic repulsion along with steric hindrance. In the crystal structure of EX7-NADPH complex, due to the Glu41Gly mutation, the 2 0 -phosphate group of the adenine ribose in NADPH can occupy the position vacated by the mutation (Fig. 2E). In addition, OP1A of NADPH is hydrogenbonded to the side-chain hydroxyls of the Ser residues ˚ ) and 45 (2.89 A ˚ ). An introduced at positions 42 (2.50 A ˚ additional hydrogen-bond (2.87 A) is formed between Ser42N and OP2A of NADPH. Hydrogen-bond shorter ˚ in length is referred as ‘‘low energy barrier than 2.5 A hydrogen-bond’’ and is known to have a covalent bondlike character [21–23]. Although the side-chain of Gln43 is not seen probably extending toward the surface, the mutation of Pro43 to Gln leaves the / and w angles unchanged, indicating that this mutation does not affect the loop structure and coenzyme specificity. The side-chain of Arg44, which is presumed to interact with the 2 0 -phosphate of NADPH, is also almost invisible in the electron density map although density of the Cb of this residue is observed. Phe46 is well defined in the hydrophobic region like the Met in the wild-type tMDH. As for Gln47, although most part of the side-chain is also well defined, the terminal amide and carbonyl groups are invisible in

Q47

B

Q47

R44

R44

44(83) 45(84)

47(86)

43(82)

S42

S42

S45

46(85)

S45 42(81)

F46

G41

F46

G41

41(80)

Fig. 1. Structure of altered loop region. (A) Stereoview of the jFoj  jFcj omit maps of the altered loop region of EX7-NADPH complex. Contour level of each map is 2.0 r. (B) Comparison of the main chain Ca trace of the tMDH-NADPH (green), EX7-NADPH (cyan), EX7-NADH (magenta), and chMDH-NADPH (yellow).

H186

A

H186

B

N130

C

D

D158

H186

E

D197

H225

D158

NADPH

N169

NADPH

F H186

D158

H186

N130 NADH

N130

N130

NADH

N130

I15

I15

I15

I15

I15

M53 Q150

Q14 G13

Q111

Q14 G13

Q14 G13

G52

Q14

G49 S45

S42

E41

S84

S81

S45

S42

S45

S42

Fig. 2. Coenzyme bound to EX7. jFoj  jFcj omit maps of the coenzymes of EX7-NADPH complex (A) and EX7-NADH complex (B). Contour level of each map is 2.0 r. Coenzyme binding sites of tMDH-NADH complex (C), chMDH-complex (D), EX7-NADPH complex (E), and EX7-NADH complex (F).

T. Tomita et al. / Biochemical and Biophysical Research Communications 347 (2006) 502–508

A

3.07

S45

B

S42

S84

2.86

S45 S84

C

S81

S42 S81

3.34 3.08

3.42 2.89

3.02

2.87 2.50

3.27 2.90 3.04

A11

w223

w176

3.01

w176 2.86 A12

505

3.22

2.7 0

A51

w223

2.98

2.99 2.88

2.84 3.36

Q111 A50

w153

G10

3.39

Q111 Q150

Q150 A11 A50

G49

NADPH

A12 A51

NADPH

w153

G10 G49 NADPH NADPH

Fig. 3. Comparison of the position of the 2 0 -phosphate group of NADPH between EX7-NADPH complex (cyan) and chMDH-NADPH complex (yellow). (A) EX7-NADPH complex, (B) chMDH-NADPH complex, and (C) superimpose of both complex.

the map, probably extending toward the surface. Phe46 and Gln47 in EX7 are located far from the 2 0 -phosphate binding site and thus have no direct interaction with NADPH. Unexpectedly, the adenine moiety is not found in the electron density map (Fig. 2A), suggesting the moiety is not recognized by EX7 tightly. In addition to the Ser residues hydrogen-bonded to the 2 0 -phosphate group, three water molecules, w153, w176, and w223, that are not seen in chMDH-NADPH complex stabilize the ribose and the 2 0 -phosphate moieties (Fig. 3A). Especially, w176 plays a key role in the coenzyme recognition through the hydrogen-bond network. In the crystal structure of the EX7-NADH complex, the structure of the loop region, Gly-Ser-Glu-Arg-Ser-PheGln, is essentially the same as that of the loop region in the EX7-NADPH complex (Fig. 1B). Although electron densities of the nicotinamide, nicotinamide ribose, and pyrophosphate moieties are observed in the map, those of the adenine and adenine ribose moieties of NADH are missing in the map (Fig. 2B), suggesting that the adenine and adenine ribose moieties of NADH are not tightly recognized by EX7. In addition to the absence of some electron densities of NADH, averaged B-factor of the ˚ 2) nicotinamide and nicotinamide ribose portions (26.15 A of this coenzyme is somewhat larger than that of the ˚ 2) (Fig. 2). These observaEX7-NADPH complex (23.55 A tions suggest that the hydrogen-bond interactions between these Ser residues 42 and 45 and the 2 0 -phosphate of NADPH are the crucial factors in the high affinity (Km of 7.4 lM) of EX7 for NADPH. Involvement of Arg44 in recognition of the 2 0 -phosphate group of the adenine moiety in the EX7 mutant In the crystal structure of the EX7-NADPH complex, electron density of the side-chain of Arg44 is almost invisible, although its role in recognition of NADPH has been expected. To investigate the role of Arg44 of the EX7 mutant in recognition of the 2 0 -phosphate group, we conducted the steady-state kinetic analyses for two additional mutants EX6 and EX4 constructed in this study. The mutant EX6 has chloroplast-type amino acid residues in the same way as the EX7 mutant, but has Arg44Gln replacement in the loop region, while the EX4 mutant

has a mutation of Gln44Arg in addition to the mutations at positions 41, 42, and 45 in EX3. By the single replacement of Arg44Gln, a significant increase in Km for NADPH resulting in the decrease in kcat/Km for NADPH was observed (Table 2). On the other hand, addition of Gln44Arg to EX3 yielding EX4 caused a marked decrease in the Km for NADPH. As results, kcat/Km for NADPH was much improved in the EX4 mutant. These results clearly indicate that Arg44 is another determinant for the coenzyme specificity toward NADPH although the sidechain is invisible in the determined crystal structure of EX7. Recognition of the adenine moiety in the EX7 mutant In the crystal structure of the tMDH-NADH complex, the adenine moiety is recognized by Ile42 from one side and Gly90 from the other side through CH/p bonds as seen in many NADH-binding proteins [24]. In the NADP(H)-dependent chMDH from F. bidentis [25], Gln150Ne2 weakly hydrogen-bonds with the neighboring N1A of NADPH (Figs. 2D and 3B). In the chMDH complex, Leu79, Gly125, and Ile145 form hydrophobic pocket for accommodating the adenine moiety of NADPH, and Gly125 has CH/p bond interaction with the adenine moiety of the coenzyme. In the crystal structure of the EX7-NADPH complex, on the other hand, the electron density of the adenine moiety of NADPH is not found in the map, suggesting that the adenine moiety of NADPH is not well recognized by the EX7 mutant. To examine this hypothesis, kinetic analyses using coenzyme analogs, nicotinamide hypoxanthine dinucleotide (phosphate) (NHD(P)H), were performed. These analogs of NAD(P)H contain a different base, hypoxanthine in place of adenine. Hypoxanthine is structurally similar but has a keto group, in the position of the N6A amide group in NAD(P)H, on its non-planer base. The kinetic parameters of EX7 for NHDPH are essentially the same as those for NADPH (Table 3). In the wild-type tMDH, however, the Km value for NHDH was 15-fold larger than that for NADH, although a concomitant increase in the kcat value that may compensate for the decrease in the kcat/Km value was observed. Thus, the loss of both N6A and the aromatic planer structure affects the affinity between tMDH and nicotinamide coenzyme. These results support the idea

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Table 2 Kinetic parameters of tMDH and the mutants Enzyme

Coenzyme

Wild-type

NADH

Km (lM) NADH

NADPH

3.0 ± 0.5 42.6 ± 5.6 740.2 ± 13.6 EX3

NADH

94.5 ± 13.8 31.7 ± 4.1

NADPH

98.5 ± 1.2 5.4 ± 0.6

EX4

NADH

164.1 ± 9.2 15.0 ± 2.0

NADPH

5.1 ± 0.4 11.7 ± 2.7

EX6

NADH

160.8 ± 11.1 9.0 ± 0.9

NADPH

27.5 ± 4.2 35.3 ± 2.6

EX7

NADH

80.9 ± 25.6 31.7 ± 4.1

NADPH

kcat/Km (lM1 s1)

(122.4 ± 5.8) 146.7 ± 7.5 74.2 ± 2.5 72.7 ± 0.6 98.1 ± 6.6 104.1 ± 4.9 140.5 ± 0.7 (145.7 ± 2.0) 188.4 ± 5.9 (111.1 ± 4.0) 246.7 ± 2.1 245.4 ± 19.2 173.4 ± 6.7 (86.8 ± 2.6) 192.2 ± 11.5 184.7 ± 3.0 80.2 ± 10.9 78.2 ± 3.6 111.7 ± 6.8 112.0 ± 1.6

(61.1) 48.9 1.7 0.1 1.0 3.3 1.4 (27.0) 1.1 (12.6) 48.3 21.0 1.1 19.2 7.0 4.8 0.9 2.5 15.1 20.7

Oxaloacetate

2.4 ± 0.6

NADPH

kcat (s1)

7.4 ± 2.6 5.4 ± 0.6

Parentheses indicate apparent values because sufficient concentrations of coenzyme and substrate could not be added.

Table 3 Kinetic parameters of tMDH and EX7 Enzyme

Coenzyme

Km for coenzyme (lM)

kcat (s1)

kcat/Km (lM1 s1)

tMDH

NADH NHDH NADPH NHDPH

2.4 ± 0.6 37.5 ± 2.6 7.4 ± 2.6 7.2 ± 0.5

122.4 ± 5.7 297.5 ± 8.8 111.7 ± 6.8 70.4 ± 1.2

61.1 7.9 15.1 9.7

EX7

that the adenine moiety of NADPH is not recognized by the EX7 mutant in contrast to those of NADH in the tMDHNADH complex and NADPH in the chMDH-NADPH complex, although EX7 shows strict NADPH preference for the reaction. Discussion The overall structures of EX7-NADPH and EX7NADH complexes are essentially the same as that of the tMDH-NADH complex. The main chain atoms of the replaced loop region between b-strand B and a-helix C in the nucleotide-binding fold in EX7-NADPH and EX7NADH complexes fit well with those of the corresponding region of tMDH and chMDH from F. bidentis. These results suggest that the reasons for the specificity change must lie in the side-chain differences. O2 0 and O3 0 of the adenine ribose moiety of NADH are commonly hydrogen-bonded to glutamate or aspartate residues in NAD(H)-dependent enzymes with the Rossmann fold [26–30]. Therefore, the negatively charged residue is one of the most principal determinants for the preference for NAD(H) over NADP(H) of these enzymes. In the tMDH-NADH complex, the carboxyl group of Glu41

occupies a position close to the probable 2 0 -phosphatebinding site and prevents the phosphate from binding by electrostatic repulsion. In the EX7-NADPH complex, Glu41Gly allows the 2 0 -phosphate group of NADPH to enter the site. Furthermore, Ser42 and Ser45 in the replaced loop make tight hydrogen-bonds with the 2 0 -phosphate of NADPH and bound three water molecules stabilize the 2 0 -phosphate and ribose moieties (Fig. 3A). Similar hydrogen-bond interactions are seen in the crystal structure of the chMDH-NADPH complex from F. bidentis (Figs. 2D and 3B) [25]. In that complex, Ser81 and Ser84, which correspond to Ser42 and Ser45 of the EX7 mutant, are hydrogen-bonded to the 2 0 -phosphate of NADPH although the location of the 2 0 -phosphate group is different from that of the EX7-NADPH complex. In chMDHNADPH complex the hydrogen-bond distance is longer than that in the EX7-NADPH complex (Fig. 3B). Consis˚ 2) of tent with this, B-factor of the 2 0 -phosphate (90.65 A NADPH in chMDH is apparently higher than that of other ˚ 2) (Fig. 2D). To compenportions of the coenzyme (39.17 A sate for the weaker binding, additional hydrogen-bonds to N1A and N3A of the adenine moiety are formed in chMDH by Gln150Ne2 and Gly49N (Fig. 3B). Since the adenine moiety is missing from the map of the

T. Tomita et al. / Biochemical and Biophysical Research Communications 347 (2006) 502–508

EX7-NADPH complex, the short hydrogen-bond interactions between the two Ser residues, Ser42 and Ser45, and the 2 0 -phosphate of NADPH are important for the high affinity of EX7 for NADPH. On the other hand, in the EX7-NADH complex, the electron densities of the adenine and the adenine ribose are not observed in the map, suggesting that these moieties are not strictly recognized in the EX7-NADH complex. The low affinity of EX7 for NADH can be explained by two possible reasons: (i) the Glu41Gly mutation removes the hydrogen-bonds between the c-carboxyl group of Glu41 and the O2 0 and O3 0 atoms of the adenine ribose moiety of NADH, and (ii) Ser42 and Ser45 are located too far from the O2 0 and O3 0 atoms of the adenine ribose moiety of NADH to form hydrogenbonds. In silico simulation of the EX7-NADPH complex model based on tMDH-NADH structure suggested that the Arg residue introduced at position 44 forms an additional electrostatic interaction with the 2 0 -phosphate of NADPH to stabilize the binding (data not shown). Consistent with this, the importance of the corresponding Arg residues in NADP(H) specificity was also shown by site-directed mutagenesis in other NADP(H)-dependent dehydrogenases [31–33]. However, in the EX7-NADPH complex, the sidechain of Arg44 is not seen in the electron density map. Similarly, even in the crystal structure of chMDH from F. bidentis, the side-chain of the corresponding Arg residue is also invisible in the map [25]. Thus, the crystallographic data do not clarify the role of the Arg residues in the interaction with NADPH. Considering that there are various conformations of arginine to recognize the 2 0 -phosphate and the adenine moieties in NADPH-binding proteins as summarized by Carugo and Argos [34], it is likely that the side-chain of Arg44 is mobile even in the enzyme–coenzyme complex but contributes to the recognition of the 2 0 phosphate of NADPH in several conformations. Conclusion The present crystallographic study coupled with site-directed mutagenesis shows that strong binding of the 2 0 -phosphate group of NADPH by two Ser residues and bound water atoms, and putative electrostatic interaction of Arg44 with the 2 0 -phosphate group are the crucial determinants for the coenzyme specificity of EX7. In this mutant strict recognition of the adenine moiety is not necessarily required to show the high NADPH preference. This is supported by the kinetic analysis using a NAD(P)H analog, NHD(P)H. In the tMDH-NADH complex, binding of the adenine moiety of NADH is stabilized by hydrophobic interactions with Ile42, similar to the case of a 2,5-diketo-D-gluconic acid reductase mutant, where the adenine moiety is stabilized by the base stacking interaction with the imidazole ring of His residue [11]. We speculate that the replacement of Ile42 with Ser enables the enzyme to interact with the 2 0 -phosphate group and, at the same time, destabilizes the interaction with the adenine moiety in the EX7 mutant.

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