d -3-Hydroxybutyrate Dehydrogenase from Pseudomonas fragi: Molecular Cloning of the Enzyme Gene and Crystal Structure of the Enzyme

doi:10.1016/j.jmb.2005.10.072

J. Mol. Biol. (2006) 355, 722–733

D-3-Hydroxybutyrate

Dehydrogenase from Pseudomonas fragi: Molecular Cloning of the Enzyme Gene and Crystal Structure of the Enzyme Kiyoshi Ito*, Yoshitaka Nakajima, Emi Ichihara, Kyohei Ogawa Naoko Katayama, Kanako Nakashima and Tadashi Yoshimoto Department of Molecular Medicinal Sciences, Graduate School of Biomedical Sciences Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

The gene coding for D-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme–NADC complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and ˚ resolution. The overall structure of P. fragi HBDH, was refined to 2.0 A including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme–NADC complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NADC-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: Pseudomonas fragi; hydroxybutyrate dehydrogenase; short chain dehydrogenase/reductase family; crystal structure; substrate recognition

Introduction D-3-Hydroxybutyrate dehydrogenase (HBDH: EC 1.1.1.30) is an enzyme that reversibly oxidizes 3-hydroxybutyrate to acetoacetate using NADC as a coenzyme. Mammals metabolize acetyl-CoA, which is formed during oxidation of fatty acids, by two pathways. One is oxidation via the citric acid cycle, while the other pathway leads to acetoacetate and D-3-hydroxybutyrate, which, together with acetone, are collectively called ketone bodies. HBDH, a mitochondrial enzyme, reversibly reduces the

Abbreviations used: HBDH, D-3-hydroxybutyrate dehydrogenase; HSDH, hydroxysteroid dehydrogenase; SDR, short-chain dehydrogenase/reductase; rms, root-mean-square. E-mail address of the corresponding author: [email protected]

free acetoacetate so produced into D-3-hydroxybutyrate.1 One of the severe complications of diabetes mellitus is diabetic ketoacidosis (DKA). DKA is caused by an increase of ketone bodies and often leads to unconsciousness. In diabetes mellitus patients, particularly those with type 1, glucose cannot be absorbed and utilized by cells due to the insulin deficiency, and fatty acid metabolism is elevated to produce acetyl-CoA. This acetyl-CoA, however, cannot enter the citric acid cycle because of the loss of oxaloacetate as an intermediate in the cycle. Excess acetyl-CoA is converted into ketone bodies. Therefore, it is important to monitor ketone bodies in the blood and urine of patients with diabetes mellitus.1 HBDH was found in rat liver mitochondria,2 and has been purified from the human heart,3 bovine heart, and rat liver.4 The enzyme has the specific

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

723

Structure of D-3-Hydroxybutyrate Dehydrogenase

requirement of phosphatidylcholine for activity.3 It has also been found in many microorganisms.5,6 Among microorganisms, however, the enzyme has been cloned thus far only from Rhodobacter sp.7 In contrast to mammalian HBDH, the bacterial enzymes do not require phospholipids for their activity. These microbial enzymes have been used for the diagnostic analysis of ketone bodies for diabetes mellitus.8 HBDH belongs to the SDR family. More than 3000 primary sequences are annotated in the sequence databases if the sequences of species variants are included. The SDR family enzymes act on a great variety of compounds and are divided into several EC classes from oxidoreductases to isomerases. Moreover, among a subgroup of oxidoreductases to which most of the enzymes belong, diverse substrate specificities are found to act on a range of compounds from steroids to aliphatic alcohols. The diversity of the SDR family might be in the same range as that of the CYP P450 family.9 The amino acid sequence identity among SDRs is relatively low, roughly 15–30%. However, the three-dimensional structures of the SDR family enzymes have architectures that are quite similar to each other, except for the substrate binding loop region.10 It is of great importance to understand the strategies to recognize the substrates of SDRs. We found the HBDH in Pseudomonas fragi, cloned the enzyme gene, and expressed the enzyme in Escherichia coli. This is the first report of the crystal structure of HBDH. Its substrate recognition mechanisms are also discussed.

Results Cloning of the D-3-hydroxybutyrate dehydrogenase gene from Pseudomonas fragi Alignment of seven amino acid sequences of HBDH revealed several conserved regions. Among them, we chose two regions, one containing a VTGSTSGIGLG sequence and the other a CPGWV(R/L)TPLV sequence. Degenerate oligonucleotide primers derived from these sequences were used to amplify a 0.54 kb fragment. Sequencing of this PCR product revealed that it is a part of the HBDH gene. Southern hybridization analysis against P. fragi chromosomal DNA using this 0.54 kb fragment as a probe showed that the HBDH gene existed in a 3.1 kb EcoRI fragment. To obtain the entire coding sequence, additional primers (HBDH11s, HBDH12s, HBDH13a, and HBDH14a) were synthesized from the 0.54 kb sequence. Upstream and downstream regions were amplified by PCR and combined to construct plasmid pHBDHfull1 as described in Materials and Methods. The nucleotide sequence of the 1265 bp XhoI-SacI region was determined. There was a 780 bp open reading frame coding for a 260 amino acid residue protein. The GCC content of the sequenced region

was 69%. As reported for the Rhodobacter HBDH gene, G/C was preferentially used for the third position of codons (234 of 260 codons) and 23 codons were not used at all. The deduced amino acid sequence showed the highest identity (89.2%) with that of Bordetella pertussis HBDH (GenBank accession no. BX640418). The sequence had greater than 56% identity with those of other bacterial HBDHs, including Ralstonia eutropha (63.5%, GenBank accession no. AF145230), Pseudomonas aeruginosa (60.4%, GenBank accession no. AE004626), Azospirillum brasilense (66.5%, GenBank accession no. AF355575), Caulobacter crescentus (56.2%, GenBank accession no. AE005999), Brucella melitensis (58.8%, GenBank accession no. AE009469), and Rhodobacter (61.9%, GenBank accession no. AF037323). The alignment is shown in Figure 1. Sequence homology was also found with vertebrate HBDHs such as human HBDH (26.9%, GenBank accession no. BC005844). Expression of HBDH For expression in E. coli, the plasmid pHBDH11 was constructed as described in Materials and Methods. Under the control of the tac promoter, the recombinant HBDH was expressed very efficiently: the activity produced per 1 ml of culture increased from 1.5 unit/ml culture in P. fragi to 134 unit/ml culture in E. coli DH5a (pHBDH11). SDS-PAGE analysis showed a subunit molecular mass of 27 kDa, which is in good agreement with the calculated value of 26,668 Da. The purified recombinant protein exhibited both NADC-dependent oxidation of D-3-hydroxybutyrate and NADH-dependent reduction of acetoacetate. The optimum pH was 8.5 and 7.0 for dehydrogenation and reduction, respectively. Although the enzyme showed NADC preference, 2% of the activity was detected with NADPC as a coenzyme. The gel-filtration analysis on a Superdex 200 gave a molecular mass of approximately 120 kDa, suggesting that the HBDH exists as a homotetramer. Substrate specificity To clarify the substrate specificity, we examined the activity of P. fragi HBDH against some b-hydroxy acids, including L-3-hydroxybutyrate, L-threonine, D-threonine, L-serine, D-serine, and DL-glycerate substrates. Only the D-enantiomer (3R configuration) of 3-hydroxybutyrate was oxidized. L-Threonine was a poor substrate, and L-serine was not a substrate. Table 1 summarizes the substrate specificity of the HBDH. The enzyme showed no activity against a-hydroxy acids, including glycolate, DL-2-hydroxybutyrate, D-lactate, and L-lactate, or against secondary alcohols, such as 2-propanol and 2-butanol. These results indicated that the enzyme recognizes the 3R-hydroxy acid structure and the 3-methyl moiety.

724

Structure of D-3-Hydroxybutyrate Dehydrogenase

Figure 1. Alignment of the four bacterial and human HBDH sequences. The secondary structure elements of P. fragi HBDH are indicated at the top of the sequences; continuous lines and double lines indicate b-strands and a-helices, respectively. Identical residues among all and the four bacterial HBDHs are shown against a black background.

Inhibitors of HBDH Since the enzyme required the strict configuration of a 3R-hydroxy acid, we tested the abilities of substrate analogs to inhibit the enzyme activity. The L-enantiomer of 3-hydroxybutyrate inhibited the enzyme activity with a Ki value of 1.6 mM, which was comparable to the Km value of D-3-hydroxybutyrate. DL-2-Hydroxybutyrate and DL-lactate were also effective inhibitors, showing Ki values of 0.81 mM and 0.90 mM, respectively (Table 2).

Interestingly, sodium cacodylate strongly inhibited enzyme activity, as described later. To clarify the substrate recognition mechanism, we solved the crystal structures of P. fragi HBDH. Qualities of the structures The final model of a ligand-free HBDH contains two subunits, each containing 260 amino acid residues, a magnesium ion, and a cacodylate anion, and 429 water molecules, with an Rfactor of

725

Structure of D-3-Hydroxybutyrate Dehydrogenase

Table 1. Substrate specificity Substrates D-3-Hydroxybutyrate

Km (mM) 1.2

L-3-Hydroxybutyrate L-Threonine

kcat/Km (sK1 mMK1)

285

237

9.1

0.023

No activity 388

D-Threonine

No activity No activity No activity No activity No activity No activity No activity

L-Serine D-Serine DL-Glycerate Glycolate DL-2-Hydroxybutyrate DL-Lactate NADC NADPC

kcat (sK1)

0.13 7.4

Activity was measured at pH 8.5 as described in Materials and Methods. Km for NADC and NADPC were determined with 25 mM D-3hydroxybutyrate.

˚ resolution (Table 3). The average 21.6% at 2.0 A thermal factors of the main chain atoms in subunits ˚ 2, respectively. Analysis ˚ 2 and 21 A A and B are 19 A of the stereochemistry with PROCHECK11 showed that all of the main chain atoms fall within the generously allowed region of the Ramachandran plot, with 400 residues in the most favored region, 43 residues in the additionally allowed region, and one residue in the generously allowed region. The enzyme–NADC complex contains 236 amino acid residues, an NADC, a magnesium ion, a cacodylate anion, and 307 water molecules, with an Rfactor of ˚ resolution. The model lacks 24 19.5% at 1.52 A residues (Pro191–Glu214), and the average thermal ˚ 2. Analysis of the factor of main chain atoms is 14 A stereochemistry with PROCHECK showed that 175 and 22 residues fall within the most favored and the additionally allowed regions of the Ramachandran plot, respectively. One residue was in the generously allowed region. Structure diagrams were drawn with the MOLSCRIPT,12 POVscriptC,13 and Raster3D14 programs. Overall and subunit structures The tetramer of ligand-free HBDH is shown in Figure 2. This oligomeric structure corresponds to a natural state in solution, as the enzyme was suggested to be a homotetramer by gel-filtration. Two subunits in the asymmetric unit (colored in cyan and orange) can be mutually superimposed ˚ for a-carbon atoms. with an rms deviation of 0.11 A This is a dimer related to each other by a noncrystallographic 2-fold axis. The HBDH tetramer shows 222-point group symmetry, and the three perpendicular axes in tetrameric SDR enzymes are conventionally named P, Q, and R.15 In the crystal, two dimers are mutually related by the [001] crystallographic 2-fold rotation axis. The Q-axis of the three non-crystallographic 2-fold axes coincides with the crystallographic 2-fold axis. The subunit structure is shown in Figure 3. The Ca atoms between ligand-free HBDH and the enzyme–NADC complex fit within an rms devi˚ with a maximum displacement of ation of 0.15 A

˚ on Leu215. The two subunit structures are 1.44 A identical except for the disordered region in the complex with NADC. The subunit has a single a/b domain that comprises a core b-sheet constituted by seven parallel b-strands (bC, bB, bA, bD, bE, bF, and bG) with three a-helices (aB, aC, and aG or aD, aE, and aF) on either side of the sheet and two a-helices (a1 and a2) protruding from the main body. The regions bA-bC and bD-bF each have a typical Rossmann fold, which is a nucleotide-binding motif, that associates an NADC or NADH. The region from bD to bG is involved in quaternary structure association and substrate binding. The regions aE-F and aG-bG, including connecting loops participate in subunit interactions on the QR and PQ-faces, respectively. The loop between bF and aG, which includes a1 and a2, constitutes a substrate-binding loop. Many three-dimensional structures belonging to the SDR family have been solved by X-ray crystallography.9 Several enzymes have sequence homology with P. fragi HBDH. The sequence identities with P. fragi HBDH for the following are 30.8, 27.3, 26.9, 25.4, 24.5, and 22.3%, respectively: 3a, 20b-hydroxysteroid dehydrogenase complexed with NADC(3a, 20b-HSDH) from Streptomyces hydrogenans (PDB code 2hsd); 16 tropinone reductase-I complexed with NADPC from Datura stramonium (PDB code 1ae1);17 sorbitol dehydrogenase from Rhodobacter sphaeroides (PDB code 1k2w); glucose dehydrogenase (29) from Bacillus megaterium (PDB code 1gco);18 3b/17b-hydroxysteroid dehydrogenase from Comamonas testosteroni (PDB code 1hxh);19 and 7a-hydroxysteroid dehydrogenase complexed with NADC(7a-HSDH) Table 2. Inhibition by substrate analogs Inhibitors L-3-Hydroxybutyrate DL-2-Hydroxybutyrate DL-Lactate Malonate Dimethyl malonate Cacodylate

Ki (mM) 1.6 0.81 0.9 3.8 80 5.6

726

Structure of D-3-Hydroxybutyrate Dehydrogenase

Table 3. Data collection, MAD and refinement statistics

Data collection Space group Cell parameters ˚) a (A ˚) b (A ˚) c (A Wavelength Resolution ˚) range (A Number of unique reflections Completeness (%) Rmerge Ranom Mean I/s(I) MAD Resolution ˚) range (A Number of derivative sites Figure of merit Refinement Resolution ˚) range (A R-factor Rfree Root-mean-square deviation ˚) Bond lengths (A Bond angles (deg.) ˚ 2) Average B-factor (A Protein chain Water molecules NADC Magnesium Cacodylate

Ligand-free

NADC complex

Peak

Edge

Remote_1

Remote_2

P21212

I222

P21212

)

)

)

64.34 99.03 110.2 1.000 30–2.00 (2.11–2.00)

64.77 99.97 109.6 1.000 40–1.52 (1.57–1.52)

64.66 99.05 110.3 0.97986 30–1.90 (2.00–1.90)

) ) ) 0.98012 )

) ) ) 0.96400 )

) ) ) 1.0000 )

47,802 (6880)

54,355(4918)

56,539(8137)

56,589(8150)

56,543(8138)

56,545(8138)

99.1 (99.2) 0.078 (0.306)

98.3 (89.7) 0.041 (0.109)

8.4 (2.4)

61.8 (21.5)

99.0 (100) 0.073 (0.226) 0.041 (0.105) 7.1 (3.0)

99.0 (100) 0.067 (0.211) 0.031 (0.091) 7.8 (3.3)

99.9 (100) 0.074 (0.247) 0.037 (0.108) 7.1 (2.8)

99.9 (100) 0.073 (0.248) 0.032 (0.105) 7.1 (2.8)

30–2.00 6 0.432 20–2.00

30–1.52

0.216 0.247

0.195 0.213

0.005 1.188

0.004 1.240

19.0 28.5 – 1.4 29.7

17.0 30.6 18.9 2.2 17.8

Ranom Z SjhICiKhIKij=SðhICiC hIKiÞ, where hI Ci and hIKi are mean intensity values of each mate in a Bijvoet pair. P P P P Rmerge Z hkl i jIhkl;i KhIhkl ij= hkl i Ihkl;i , where IZobserved intensity and hIiZaverage intensity for multiple measurements. Values in parentheses refer to the last resolution shell.

from E. coli (PDB code 1ahh). 20 The subunit structures of these six enzymes have essentially the same topology and were superimposed onto the ligand-free HBDH by a least-squares method.

As shown in Figure 4, these structures are similar to each other, and the corresponding Ca atoms (except for the substrate-binding loop) fit within ˚ . However, the subunit rms deviations of 0.87–1.04 A

Figure 2. Ribbon diagram of ligand-free HBDH. The tetramer is viewed along P-(right) and R-(left) axes of the noncrystallographic 2-fold axes consisting of 222 symmetry. Each subunit is shown in a different color. The Q-axis coincides with the crystallographic 2-fold axis. The dimeric structure of ligand-free HBDH (colored in cyan and orange) is in an asymmetric unit.

Structure of D-3-Hydroxybutyrate Dehydrogenase

727

Figure 3. The subunit structures of ligand-free HBDH and enzyme– NAD C complex. Ligand-free HBDH with secondary structure assignments and enzyme–NADC complex are shown by ribbon models. The cacodylate anions and NADC are indicated by balland-stick models.

structure of HBDH differs in the loop between bB and aC because the loop has an insertion at Asp38. Consequently, the N-terminal portion of the aC helix is pushed further away to the solvent area unlike the case with the other members. The strictly conserved residues of the catalytic tetrad in the SDR family are Asn114, Ser142, Tyr155, and Lys159 in P. fragi HBDH. The positions of these four residues conform to those in fitted SDR enzymes. In addition, a water molecule is found hydrogen bonding to Asn114 and Lys159, suggesting a proton relay network proposed by Filling et al.21 The NADC binding site The NADC binding site and scheme diagram are shown in Figure 5. The electron density corresponding to NADC is well defined. The NADC binding mode is similar to those found in known structures of the SDR family. An adenine ring of NADC is accommodated in the hydrophobic pocket comprising Gly11, Phe36, Leu64, Ala90, ILe93, and Leu113. The amino group of the adenine ring makes a hydrogen bond with Asp63. The hydroxyl groups of adenosine ribose are not

involved in the hydrogen bond with the aspartate residue revealed in 3a, 20b-HSDH and 7a-HSDH. 16,20 O2A and O3A form hydrogen bonds to the main chain NH of Phe36 and the hydroxyl group of Thr13, respectively. The hydrogen bonding to threonine is quite similar to that in tropinone reductase-I or glucose dehydrogenase,17,18 whereas the hydrogen bonding to the main chain NH is unique. The loop of Gly11-Gly17 between bA and aB has a typical sequence of G-x-xx-G-x-G, and it recognizes the pyrophosphate moiety with a hydrogen bond to the main chain NH of Ile16. The nicotinamide nucleoside moiety is accommodated in the hydrophobic pocket composed of Ile16, Ile140, Pro185, and Val188, and the hydroxyl groups or the nicotinamide ring forms a hydrogen bond with the main chain carbonyl group of Asn90, Tyr155, and Lys159 or with the main chain NH of Val188, respectively. The sidechain of Thr190, rotated about 458 from the position found in the ligand-free enzyme, forms hydrogen bonds to the nicotinamide NH2 group and to the phosphate of NADC. The recognition of NADC by Thr190 is considered to be a significant factor in the conformational change of the substrate-binding loop.

Figure 4. The stereo diagram of SDRs superimposed onto ligand-free HBDH. The Ca trace of HBDH is shown by the orange line, and 2hsd, 1ae1, 1k2w, 1gco, 1hxh, and 1ahh by red, blue, cyan, blue-violet, magenta, and green lines, respectively.

728

Structure of D-3-Hydroxybutyrate Dehydrogenase

Figure 5. Drawing of the NADC binding site in HBDH. (a) The wire frame shows the FoKFc omit map for NADC and cacodylate anion contoured at 2s levels. The stick models show residues interacting with NADC or cacodylate anion. (b) The scheme diagram shows hydrogen-bonding interaction with NADC.

Inhibition by cacodylate The reactions of many NAD(P)C-dependent dehydrogenases follow a sequential mechanism, in which the coenzyme binding precedes the substrate binding. It is generally accepted that the SDR enzymes show a large conformational change upon substrate binding. However, the binding of the nucleotide cofactor alone does not usually induce a significant conformational change. Moreover, the structures of the substrate-binding loop in many SDR enzymes are often disordered in the absence of a bound cofactor and substrate.10,22 Interestingly, the corresponding region in the ligand-free HBDH (a 1Ka 2 ) shows a defined electron density, and the conformation of this region is changed in the enzyme–NAD C complex. A cacodylate anion is assigned in the electron density map of the putative substrate-binding region in both the ligand-free enzyme and the enzyme–NADC complex. A comparison of the structures of cacodylate and the substrate D-3-hydroxybutyrate reveals that they have similar methyl and oxo groups. Then we tested the ability of sodium cacodylate to inhibit HBDH activity. As shown in Table 2, cacodylate is a competitive inhibitor of D-3-hydroxybutyrate, having a Ki value of 5.6 mM. We concluded that the cacodylate anion, a constituent in the reservoir buffer, acts as a substrate analog during crystal formation. This should be the reason why the substrate-binding loop moved in the enzyme–NADC complex. Binding of cacodylate The binding mode of cacodylate is shown in Figure 6. The cacodylate anion is tightly bound to the active site in the ligand-free enzyme (enzymecacodylate). One of the methyl groups of cacodylate binds to a hydrophobic pocket consisting of Ala143,

His144, Gly186, Trp187, and Trp257. One of the oxygen atoms forms hydrogen bonds with Ser142 ˚ and 2.55 A ˚, and Tyr155 at distances of 2.58 A respectively, while in the enzyme–NADC complex

Figure 6. Close-up view of the cacodylate-binding site. Ribbon and stick model of the active site of the enzyme– NADC complex is superimposed onto that of the ligandfree HBDH. The residues and hetero molecules of ligandfree HBDH are shown in cyan, and those of enzyme– NADC complex are in CPK colors. In both structures, a methyl group of the cacodylate anion is accommodated in the hydrophobic pocket composed of Ala143, Trp187, and Trp257, and a cacodylate oxygen atom forms a hydrogen bond to Ser142 and Tyr155. Two cacodylate anions, however, are bound to different positions. The distance ˚ . Another methyl between two arsenic atoms is 1.71 A group of the cacodylate anion in the ligand-free HBDH is close to the 4-position of nicotinamide ring in the ˚. enzyme–NADC complex, at a distance of 1.01 A

Structure of D-3-Hydroxybutyrate Dehydrogenase

(enzyme–NADC-cacodylate), the binding of the cacodylate methyl group to the hydrophobic pocket and the hydrogen bonding to Ser142 and Tyr155 ˚ and 2.59 A ˚ , respectively) are similar to those (2.52 A found in the ligand-free enzyme. However, the ˚ from the position in cacodylate anion moves 1.71 A the ligand-free enzyme, and the cacodylate’s negative charge is recognized by the amino groups of Lys152 and His144, to which the distances are ˚ and 2.65 A ˚ , respectively. 2.99 A

Discussion The gene encoding HBDH was cloned from the chromosomal DNA of P. fragi and expressed in E. coli. The recombinant enzyme was purified to homogeneity and crystallized. Using these crystals along with those of the SeMet-substituted enzyme, the crystal structures of both the ligand-free HBDH and the enzyme–NADC complex were solved for the first time. From the structures of the ligand-free enyme and the enzyme–NADC complex, the substrate recognition mechanism can be predicted as shown in Figure 7. In the ligand-free enzyme, the electron density map of the indole six-membered ring of Trp187, which is a major constituent of the hydrophobic pocket, is slightly obscure compared with that of the five-membered ring. This indicates that the side-chain rotates around an axis of Cb–Cg. On the other hand, the side-chain of Trp187 in the enzyme–NADC complex is fixed and in a different position from that in the ligand-free enzyme. A large movement of Trp187, an approximately 388 rotation of the side-chain, must occur upon substrate binding (Figure 6). In the enzyme–NADC complex, the distance from the C4 position of

Figure 7. A possible substrate-binding mode. The D-3hydroxybutyrate is shown in red. Residues involved in the recognition of the substrate carboxyl group are colored in blue (Gln94, His144, and Lys152).

729 the nicotinamide ring to the arsenic atom is ˚ . If the chiral carbon atom of D-3-hydroxybu3.85 A tyrate is placed in the position of the arsenic atom, the substrate is located too far away to transfer the hydride to nicotinamide. Considering that the distance from the corresponding C4 position is ˚ in the ligand-free enzyme, it seems very 2.16 A likely that the cacodylate position in the ligand-free enzyme actually represents the substrate-binding site. On the basis of this position in the ligandfree enzyme, the D -3-hydroxybutyrate model was applied. Consequently, one of the substrate carboxyl oxygen atoms overlapped with the cacodylate oxygen atom binding to His144 and Lsy152, and the other was able to interact with Gln94. From this model, it is presumed that the substrate carboxyl group is recognized by Gln94, His144, and Lys152. These interactions direct the methyl and carboxyl groups of the substrate in parallel, and then the 3R-hydroxyl group should be oriented to the catalytic tetrad of Asn114-Ser142Tyr155-Lys159. These residues are strictly conserved among the amino acid sequences of bacterial HBDHs. It is considered that HBDH changes from the open to the closed form upon substrate binding, as do other members of the SDR family. A closed model of HBDH was established by moving a whole region of the substrate-binding loop based on the conformations of Thr190 and Leu215 in the enzyme– NADC complex. From the model, it was shown that Gln196 approached the substrate carboxyl recognition site, and the hydrophobic residues of Leu192 and Val193 covered the active site as if they were displacing the solvents. These residues belonging to the a1-helix would be involved in substrate recognition and catalytic reaction. HBDH belongs to the SDR family. HBDH’s sequence identities with several other enzymes in that family are in the range of 15–30%. The identical regions include a T-G-x-x-x-G-x-G (residues 10–18) coenzyme binding motif, N-N-A-G (89–92) stabilizing the central b-sheet, the region from bE to the N-terminal half of the aF containing the catalytic Ser-Tyr-Lys triad, and residues involved in substrate binding.9 We have confirmed that the seven hypothetical HBDHs deposited in the DNA databases are truly the genes for HBDH, because more than 56% identity is revealed with the P. fragi enzyme, which is experimentally proven to catalyze the given reaction. The identical residues include almost all of those involved in substrate specificity. Variations are found mainly in the regions of aC-bC-aD, a1- a2 and bG. The aC-bC-aD region faces the solvent area and is involved in neither the quaternary structure association nor substrate binding. Although the sequence of bG differs greatly, bG should be important in interaction between the subunits through the formation of the b-sheet. This is also the case for the aG region, in which inter-subunit contact in this region is evident despite a relatively low sequence identity. The region of a1-a2, constituting a substrate-binding loop, also shows a large sequence variation.

730 However, the connecting region for bF and a1 containing Thr190 is strictly conserved among HBDHs. This is consistent with the fact that the loop between bF and a1 is very important for conformational change of the substrate-binding loop and the catalytic reaction. This threonine residue is well conserved in other SDR enzymes. The site-directed mutagenesis of Thr188 of human 15-hydroxyprostaglandin dehydrogenase has been reported. 23 While the T188S mutant retained substantial activity, the T188A and T188Y mutants lost their activity, indicating that the hydroxyl group of the threonine residue might be involved in the interactions of NADC and the substrate leading to the conformational change. In P. fragi HBDH, little NADPC activity was detected; the Km values for NADC and NADPC were 0.12 mM and 7.4 mM, respectively. According to the coenzyme-based functional assignments,24 classical SDRs, preferring NAD(H) over NADP(H), have an acidic residue at the end of the second b-sheet (position 36, 37, or 38). There are no acidic residues in the corresponding positions of P. fragi HBDH. bB of the P. fragi HBDH ends with Asn34 and is followed by the loop consisting of the G-F-G-D sequence. This loop recognizes an adenine portion of NADC between bB and aC. The loop conformation is distinct from other members of the SDR family, and the main chain N atom of Phe36 forms a unique hydrogen bond to O3A of the NADC adenosine ribose. A steric hindrance is likely to exist between Phe36 and the 3-phosphate group of NADPC, and this hindrance would explain the NADC preference. The little activity of NADPC can be explained by the presence of two Gly residues in the loop, because the steric hindrance might be somewhat dissolved by a conformational change. In the present structure of the ligand-free enzyme, the electron density map for the substrate-binding loop can be observed clearly. It corresponds to a so-called open conformation of SDRs. A cacodylate anion is evident at the active site in the absence of NADC. The enzyme remains in the open conformation in the ligand-free enzyme; this accommodates only a cacodylate anion, which is a substrate analog inhibitor. It is accepted that the substrate-binding loop moves in order to form more contacts to the substrate and thus to form a closed conformation upon substrate binding. The presence of a nucleotide cofactor is probably necessary for this conformational change to occur. In the presence of NADC, a probable closed conformation structure is formed. However, the open-to-closed conformational change does not complete with a substrate analog inhibitor of cacodylate. Accordingly, 24 residues (Pro191—Glu214) are missing in the refined structure of the enzyme–NADC complex. The sequence identity of P. fragi HBDH with mammalian HBDHs was approximately 23%. The catalytic tetrad is completely conserved in both P. fragi and human HBDH and readily aligned. The major difference between them is the length of

Structure of D-3-Hydroxybutyrate Dehydrogenase

the polypeptide chain, because of the mitochondrial targeting sequence at the N terminus and the phospholipid-binding region at the C terminus in the human HBDH.25 In contrast to the conservation of the catalytic tetrad, other critical residues claimed in the present study are not well conserved. A residue corresponding to Trp257 can be found in human HBDH, and an arginine is present at the position of Lys152, which interacts with the carboxyl group of the substrate in P. fragi HBDH. Other residues are not readily aligned. This may suggest that the substrate-binding pocket in human HBDH may consist of a combination of phospholipids and the substrate-binding region of the enzyme. The structure of mammalian HBDH has to be solved before we can understand the phospholipid requirement in detail. In conclusion, the crystal structure of P. fragi HBDH has been determined. The substrate recognition mechanism is revealed by the interaction between the enzyme on the one hand and the methyl and carboxylate groups of the substrate on the other. A site-directed mutagenesis study is underway to confirm this proposal and to further elucidate the substrate recognition and catalytic mechanisms of HBDH.

Materials and Methods Materials Restriction endonucleases and other DNA modification enzymes were purchased from TaKaRa Shuzo Co. The oligonucleotide primers were synthesized by Sigma Genosys. The BigDye terminator cycle sequencing kit and other reagents used for sequencing were obtained from Applied Biosystems. 3-D(K)(3R)-Hydroxybutyric acid sodium salt, 3-L(C)(3S)-hydroxybutyric acid sodium salt, and D-threonine were purchased from SigmaAldrich. Phenazine methosulfate (PMS), nitro blue tetrazolium (NBT), and Triton X-100 were products of Nacalai Tesque. NADC was from Kojin Kagaku. Polyethylene glycol, which was used for crystallization and had a mean molecular mass of 3000 (PEG3000), was purchased from Hampton Research. Seleno-L-methionine was from Sigma-Aldrich. L-Threonine, L-serine, and all other chemicals were of the highest grade available from Nacalai Tesque or Wako Pure Chemical Industries. Bacterial strains and plasmids E. coli DH5a (FK, f80dlacZDM15, D(lacZYA-argF)U169, recA1, endA1, gyrA96, thiK1, supE44, relA1, hsdR17(rK, mC), deoR, phoA) was used for DNA manipulation and protein expression. SeMet-substituted enzyme was expressed in E. coli DL41 (FK, metA28). pGEM-T-easy (Promega) was used for cloning PCR products, and pKK223-3 (Pharmacia) was used for protein expression. Cloning of the HBDH gene DNA manipulations were performed following standard protocols26 Oligonucleotide primers were designed from homologous regions of the alignment of amino acid

731

Structure of D-3-Hydroxybutyrate Dehydrogenase

sequences deduced from several bacterial genomic sequences assumed to encode HBDH (B. pertussis, Ralstonia eutropha, P. aeruginosa, A. brasilense, C. crescentus, B. melitensis, and Rhodobacter sp.: GenBank accession nos. BX640418, AF145230, AE004626, AF355575, AE005999, AE009469, and AF037323, respectively). A DNA fragment of the P. fragi HBDH gene was amplified by PCR using primers HBDH1s: 5 0 -GGTTCTACCAGCGGNATHGG-3 0 , HBDH5a: 5 0 -GTCAGCACCCAKCCKGGRCA-3 0 . The nucleotide sequence of the resultant 0.54 kb fragment was determined. Using this sequence, additional primer sets were synthesized to amplify both upstream and downstream parts of the HBDH gene, using the In Vitro Cloning kit (TaKaRa Shuzo). Briefly, P. fragi DNA was digested with EcoRI, and the EcoRI cassette was ligated at both ends. This DNA was used as a template to amplify the remainder of the HBDH gene with cassette primers C1 and C2 and primers 11s, 12s, 13a, and 14a complementary to the 0.54 kb PCR product. The nucleotide sequence was determined by the ABI Prism 3100 Avant Genetic Analyzer using the BigDye terminator v1.1 cycle sequencing kit. The upstream part (0.8 kb) and the downstream part (2.7 kb) were cloned in pGEM-T-easy to produce pHBDHUP1 and pHBDHDOWN1, respectively. To obtain a DNA fragment containing the entire structural gene, plasmid pHBDHDOWN1 was digested with NcoI and SphI, and the 1.7 kb fragment was ligated into the same restriction sites of pHBDHUP1 to produce pHBDHfull1. pHBDHfull2, containing a 1.35 kb fragment, was constructed by deleting a 1.1 kb downstream ApaI fragment from pHBDHfull1. In the HBDH structural gene, a DNA fragment containing a unique EcoRI recognition site adjacent to the initiation codon was amplified by PCR using upstream primer HBDHATG15 (5 0 -CCCGAATTCATG CTCAAAGGAAAAGTCGC-3 0 ), downstream primer HIII-T7 (5 0 -GCCCAAGCTTTGTAATACGACTCACTA TAG-3 0 ), and the plasmid pHBDHfull2 as a template. The resultant 0.99 kb fragment was digested with EcoRI and HindIII, and ligated into the similarly digested pKK223-3 to give plasmid pHBDH11. The nucleotide sequence of the inserted fragment was determined as described above, and it will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the GenBank accession no. AB183516. Assay of hydroxybutyrate dehydrogenase activity The reaction mixture contained 100 mM Tris–HCl (pH 8.5), 25 mM D-3-hydroxybutyrate sodium salt, 2 mM NADC, and a varying amount of enzyme. After 3 min pre-incubation at 37 8C, the reaction was started by adding 0.05 ml of enzyme solution and monitored by measuring absorbance at 340 nm for 2 min. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the reduction of 1 mmol of NADC per minute under the conditions described. Alternatively, the activity was assayed by diformazan formation. The reaction mixture (1.0 ml), containing 100 mM Tris–HCl (pH 8.5), 0.2% Triton X-100, 25 mM D-3-hydroxybutyrate sodium salt, 2 mM NADC, 0.001% PMS, 0.01% NBT, and a varying amount of enzyme, was incubated for 15 min at 37 8C. The reaction was stopped by the addition of 3 ml of 0.3 M HCl. Diformazan formed via the NADH-PMS-NBT coupling reaction was spectrophotometrically measured at 570 nm. One unit of enzyme activity was defined as the amount of enzyme that formed 0.5 mmol of diformazan per minute under the above conditions.

Purification of the enzymes The recombinant enzyme was purified as described.27 Briefly, crude cell-free lysate derived from E. coli DH5a (pHBDH11) was made to 45% saturated ammonium sulfate and the enzyme remaining in the clear supernatant was fractionated by a hydrophobic interaction chromatography on a Toyopearl HW65C column (Tosoh, Japan). Then active fractions were concentrated by ammonium sulfate-precipitation at 70% saturation. The enzyme was further purified by an ion-exchange column chromatography on a Toyopearl SuperQ-650C (Tosoh). Elution by a linear gradient from 0 mM to 500 mM NaCl yielded a homogeneous enzyme preparation as judged by SDS-PAGE. The pure enzyme solution was concentrated to 20 mg/ml using a Centricon 10 (Millipore) and stored at K80 8C after dialysis against 20 mM Tris–HCl buffer (pH 7.5). Expression and purification of SeMet-substituted enzyme The expression plasmid pHBDH11 was introduced into E. coli DL41.28 The synthetic LeMaster medium (5 ml) was inoculated with a single colony of E. coli DL41 (pHBDH11) and incubated at 37 8C overnight with shaking. Then 1250 ml of the same LeMaster medium was inoculated with 5 ml of the overnight preculture. After cultivation at 37 8C for 18 h with shaking, the cells were suspended in 20 mM Tris–HCl buffer (pH 7.5), and the SeMet-substituted enzyme was purified and crystallized in the manner described for the unsubstituted enzyme. Crystallization and data collection The crystallization of ligand-free HBDH with preliminary crystallographic analysis has been reported.27 The crystallization for native and selenomethionyl HBDH was carried out by essentially the same method using the same reservoir solution (12% (w/v) PEG3000, 100 mM sodium cacodylate buffer (pH 6.5), 200 mM MgCl2). Crystals belonged to space group P21212 with the ˚. following cell dimensions: aZ64.3, bZ99.0, cZ110.2 A Two subunits of tetramer are found in an asymmetric unit, and 63% of the crystal volume is occupied by solvent. The crystallization for native HBDH complexed with NADC (enzyme–NADC complex) was performed under the same conditions as native crystals, using 10.8 mg/ml protein solution containing 3 mM NADC. The crystals of the enzyme–NADC complex belonged to space group I222 with cell parameters of aZ64.8, bZ ˚ , and a subunit was found in an 100.0, and cZ109.6 A asymmetric unit. The native and enzyme–NADC complex data were collected at 100 K with an ADSC Quantum 4R CCD detector system, using a wavelength ˚ from the synchrotron radiation source at the of 1.0000 A Photon Factory BL6A station (Tsukuba, Japan). In a similar fashion, the selenomethionyl data set was collected using wavelengths of 0.96400, 0.97986, ˚ at the Spring-8 BL38B1 station 0.98012, and 1.0000 A (Hyogo, Japan). Data collection under cryogenic conditions was performed as described. All data sets were processed and scaled using MOSFLM29 and SCALA from the CCP4 suite30 or HKL200031 (Table 3). Since the value for mean I/s(I) for the NADC–enzyme complex was very high, we tried to collect higher resolution data. However, the present data were the best obtained.

732 Structure determination and refinement HBDH belongs to the SDR family and shows sequence homology with SDRs whose structures are known. We previously tried to carry out the molecular replacement method using AMoRe 32 with several SDR family enzymes, which have 22–31% sequence identity with P. fragi HBDH, as search models. However, no solutions of either molecular orientation or molecular packing were found.27 The structure of ligand-free HBDH was solved by the multiple-wavelength anomalous diffraction (MAD) method based on the Se absorption edge. All data scaling and map calculations were performed with the programs of the CCP4 suite.30 Determination of six selenium sites, refinement of the heavy-atom parameters, and calculation of the initial phases were performed with the SOLVE program,33 resulting in a ˚ resolution. The mean figure of merit of 0.43 at 30–2.0 A map was improved by the process of solvent flattening with the RESOLVE program33 to give a mean figure of ˚ merit of 0.70. The model was gradually built into a 2.0 A resolution map through several cycles of model building using the XtalView program.34 In a Fourier map calculated using SeMet derivative data, the 24 residues from Pro191 to Glu214 were unclear among a model of 260 residues per subunit. Then the model was built in a ˚ resolution map calculated using native data, and 2.0 A was clarified. The structure of ligand-free HBDH was refined by simulated annealing and energy minimization using the ˚ to 2.0 A ˚ resolution CNS program.35 Native data from 20 A were used, and then Rfactor and Rfree were reduced to 26.9% and 28.9%, respectively. The structure was scrutinized by inspecting the composite omit map. A difference map displayed two large peaks per subunit. One peak, by Asp38, was designated magnesium ion by considering the size, peak height, and four-coordinate bond to this peak with Asp38, His128* in the neighboring molecule, and two water molecules. The other is located at the active site, which was designated a cacodylate anion by considering the shape, size, peak height, and possible interactions of this peak with neighboring amino acid residues. Water molecules were picked up on the basis of the peak height and the distance criteria from the difference map. Water molecules whose thermal factors ˚ 2 (maximum thermal factor of the amino were above 65 A chain) after refinement were removed from the list. After several rounds of refinements and manual rebuilding, Rfactor and Rfree were reduced to 22.8% and 25.7%, respectively. Further model building and refinement cycles resulted in an Rfactor of 21.6% and Rfree of 24.7%, using 47,737 reflections (Table 3). The maximum thermal ˚ 2. factor of water molecules was 55 A The initial phase of the enzyme–NADC complex was determined by the molecular replacement method, using a subunit of ligand-free HBDH as a search model. The best solution with a high correlation coefficient was found by rotation and translation function searches using the AMoRe program from the CCP4 suite.32 The same refinement procedure was applied to the enzyme– NADC complex. The Rfactor value became less than 30% by the first refinement cycle, and the difference Fourier map showed residual electron density corresponding to the bound NADC. In much the same way as was seen with the ligand-free enzyme, the two large peaks shown in the difference map were designated magnesium ion and cacodylate anion. Water molecules were picked up from the difference map, and further model building and

Structure of D-3-Hydroxybutyrate Dehydrogenase

refinement cycles resulted in Rfactor and Rfree values of 19.5% and 21.3%, respectively, calculated for 54,355 ˚ resolution range reflections observed in a 30–1.52 A (Table 3). PDB accession codes The atomic coordinates and structure factors for ligand-free enzyme and enzyme–NAD C complex structures (codes 1WMB and 1X1T, respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics†.

Acknowledgements This study was supported by the National Project on Protein Structural and Functional Analyses run by the Japanese Ministry of Education, Culture, Sports, Science and Technology.

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Structure of D-3-Hydroxybutyrate Dehydrogenase

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Edited by R. Huber (Received 1 July 2005; received in revised form 25 October 2005; accepted 26 October 2005) Available online 14 November 2005