Crystal Structure of Pseudomonas fluorescens Mannitol 2-Dehydrogenase Binary and Ternary Complexes
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m206914200
ISSN1083-351X
AutoresK.L. Kavanagh, Mario Klimacek, Bernd Nidetzky, David K. Wilson,
Tópico(s)Protein Structure and Dynamics
ResumoLong-chain mannitol dehydrogenases are secondary alcohol dehydrogenases that are of wide interest because of their involvement in metabolism and potential applications in agriculture, medicine, and industry. They differ from other alcohol and polyol dehydrogenases because they do not contain a conserved tyrosine and are not dependent on Zn2+ or other metal cofactors. The structures of the long-chain mannitol 2-dehydrogenase (54 kDa) from Pseudomonas fluorescens in a binary complex with NAD+ and ternary complex with NAD+and d-mannitol have been determined to resolutions of 1.7 and 1.8 Å and R-factors of 0.171 and 0.176, respectively. These results show an N-terminal domain that includes a typical Rossmann fold. The C-terminal domain is primarily α-helical and mediates mannitol binding. The electron lone pair of Lys-295 is steered by hydrogen-bonding interactions with the amide oxygen of Asn-300 and the main-chain carbonyl oxygen of Val-229 to act as the general base. Asn-191 and Asn-300 are involved in a web of hydrogen bonding, which precisely orients the mannitol O2 proton for abstraction. These residues also aid in stabilizing a negative charge in the intermediate state and in preventing the formation of nonproductive complexes with the substrate. The catalytic lysine may be returned to its unprotonated state using a rectifying proton tunnel driven by Glu-292 oscillating among different environments. Despite low sequence homology, the closest structural neighbors are glycerol-3-phosphate dehydrogenase,N-(1-d-carboxylethyl)-l-norvaline dehydrogenase, UDP-glucose dehydrogenase, and 6-phosphogluconate dehydrogenase, indicating a possible evolutionary relationship among these enzymes. Long-chain mannitol dehydrogenases are secondary alcohol dehydrogenases that are of wide interest because of their involvement in metabolism and potential applications in agriculture, medicine, and industry. They differ from other alcohol and polyol dehydrogenases because they do not contain a conserved tyrosine and are not dependent on Zn2+ or other metal cofactors. The structures of the long-chain mannitol 2-dehydrogenase (54 kDa) from Pseudomonas fluorescens in a binary complex with NAD+ and ternary complex with NAD+and d-mannitol have been determined to resolutions of 1.7 and 1.8 Å and R-factors of 0.171 and 0.176, respectively. These results show an N-terminal domain that includes a typical Rossmann fold. The C-terminal domain is primarily α-helical and mediates mannitol binding. The electron lone pair of Lys-295 is steered by hydrogen-bonding interactions with the amide oxygen of Asn-300 and the main-chain carbonyl oxygen of Val-229 to act as the general base. Asn-191 and Asn-300 are involved in a web of hydrogen bonding, which precisely orients the mannitol O2 proton for abstraction. These residues also aid in stabilizing a negative charge in the intermediate state and in preventing the formation of nonproductive complexes with the substrate. The catalytic lysine may be returned to its unprotonated state using a rectifying proton tunnel driven by Glu-292 oscillating among different environments. Despite low sequence homology, the closest structural neighbors are glycerol-3-phosphate dehydrogenase,N-(1-d-carboxylethyl)-l-norvaline dehydrogenase, UDP-glucose dehydrogenase, and 6-phosphogluconate dehydrogenase, indicating a possible evolutionary relationship among these enzymes. mannitol dehydrogenase 6-phosphogluconate dehydrogenase multiwavelength anomalous dispersion N-(1-d-carboxyethyl)-l-norvaline dehydrogenase glycerol-3-phosphate dehydrogenase nicotinamide adenine dinucleotide (oxidized) P. fluorescens mannitol 2-dehydrogenase short-chain dehydrogenase/reductase UDP-glucose dehydrogenase Stanford Synchrotron Radiation Laboratory Mannitol, a six-carbon non-cyclic polyol, is the most abundant sugar alcohol found in nature. Produced in plants, fungi, protozoa, and bacteria as a storage compound for carbon and reducing equivalents, it also functions in response to oxidative stress and as an osmoregulator (1Schmatz D.M. Parasitology. 1997; 114: S81-S89Crossref PubMed Google Scholar, 2Jennings D.H. Adv. Microb. Physiol. 1984; 25: 149-193Crossref PubMed Scopus (151) Google Scholar, 3Hoerer S. Stoop J. Mooibroek H. Baumann U. Sassoon J. J. Biol. Chem. 2001; 276: 27555-27561Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Jennings D.B. Ehrenshaft M. Pharr D.M. Williamson J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15129-15133Crossref PubMed Scopus (176) Google Scholar, 5Shen B. Hohmann S. Jensen R.G. Bohnert H.J. Plant Physiol. (Bethesda). 1999; 121: 45-52Crossref PubMed Scopus (163) Google Scholar). d-Mannitol is used extensively in the food and pharmaceutical industry because of its favorable bulking properties and the fact that it does not cause tooth decay and is safe for diabetics. The traditional method of industrially producing mannitol involves the reduction of fructose using a metal catalyst and hydrogen gas, resulting in nearly equal amounts of d-sorbitol andd-mannitol, which must then be separated. In general, mannitol dehydrogenases (MDH)1 catalyze the NAD(P)+-dependent reversible oxidation ofd-mannitol or d-mannitol-1-phosphate to the corresponding ketose, d-fructose, or d-fructose 6-phosphate. These secondary alcohol dehydrogenases are specific for the C2(R) configuration of polyhydroxylated compounds and are of interest because of their potential applications in chiral synthesis. More recently, mannitol dehydrogenases have been identified in plants that catalyze the oxidation of d-mannitol tod-mannose, an aldose (6Stoop J.M. Pharr D.M. Arch. Biochem. Biophys. 1992; 298: 612-619Crossref PubMed Scopus (43) Google Scholar). MDHs have been characterized from plants and fungi that are members of the medium-chain zinc-containing dehydrogenase/reductase family (7Williamson J.D. Stoop J.M.H. Massel M.O. Conkling M.A. Pharr D.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7148-7152Crossref PubMed Scopus (99) Google Scholar, 8Suvarna K. Bartiss A. Wong B. Microbiology. 2000; 146: 2705-2713Crossref PubMed Scopus (40) Google Scholar). Other MDH from fungi are members of the short-chain dehydrogenase/reductase (SDR) family (3Hoerer S. Stoop J. Mooibroek H. Baumann U. Sassoon J. J. Biol. Chem. 2001; 276: 27555-27561Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 9Niehaus Jr., W.G. Dilts Jr., R.P. J. Bacteriol. 1982; 151: 243-250Crossref PubMed Google Scholar). Often, bacterial MDHs do not share significant similarity with either of these families (10Schneider K.-H. Giffhorn F. Kaplan S. J. Gen. Microbiol. 1993; 139: 2475-2484Crossref PubMed Scopus (41) Google Scholar) but instead belong to a family of long-chain MDH that so far includes 54 recognized members. These members are classified by the Protein Families Data Base as family 01232 (11Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (1996) Google Scholar). More recent work increased this number to 66. 2Klimacek, M., Kavanagh, K., Wilson, D., and Nidetzky, B., in press. 2Klimacek, M., Kavanagh, K., Wilson, D., and Nidetzky, B., in press. Sequence identity with other long-chain dehydrogenases is low, typically around 10%. Most members of the prokaryotic long-chain MDH family have been identified by primary sequence alone (11Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (1996) Google Scholar), and a limited number of these proteins have had their specificity characterized and activity quantitated. The proteins that have been studied are monomeric long-chain dehydrogenases of a molecular mass of ∼54 kDa (10Schneider K.-H. Giffhorn F. Kaplan S. J. Gen. Microbiol. 1993; 139: 2475-2484Crossref PubMed Scopus (41) Google Scholar,13Slatner M. Nidetzky B. Kulbe K.D. Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (48) Google Scholar, 14Novotny M.J. Reizer J. Esch F. Saier Jr., M.H. J. Bacteriol. 1984; 159: 986-990Crossref PubMed Google Scholar). This family so far includes mannitol 2-dehydrogenases, mannitol-1-phosphate 5-dehydrogenases, d-mannonate dehydrogenases, sorbitol dehydrogenases,l-sorbose reductase, fructuronate reductase, altronate oxidoreductases, and d-arabinitol dehydrogenases. In addition to their significance as an alcohol dehydrogenase that employs a novel mechanism, interest in long-chain MDHs originates from several potential uses: (i) transgenic expression of bacterial MDHs in plants has been tested to improve salt tolerance and resistance to oxidative stress in agricultural crops (15Tarczynski M.C. Jensen R.G. Bohnert H.J. Science. 1993; 259: 508-510Crossref PubMed Scopus (530) Google Scholar), (ii) quantitative analysis of mannitol concentration in serum and urine via a simple and sensitive enzymatic assay has potential clinical use (16Diamandis E.P. Grass C.L. Uldall R. Mendelssohn D. Maini D. Clin. Biochem. 1992; 25: 457-462Crossref PubMed Scopus (7) Google Scholar), and (iii) enzymatic production of d-mannitol from fructose would reduce downstream purification (17Slatner M. Nagl G. Haltrich D. Kulbe K.D. Nidetzky B. Biocat. Biotrans. 1998; 16: 351-363Crossref Scopus (33) Google Scholar). An inducible mannitol 2-dehydrogenase belonging to the long-chain MDH family was isolated from Pseudomonas fluorescens DSM50106 (pfMDH, EC 1.1.1.67) (18Brunker P. Altenbuchner J. Kulbe K.D. Mattes R. Biochim. Biophys. Acta. 1997; 1351: 157-167Crossref PubMed Scopus (49) Google Scholar). It catalyzes the reversible oxidation ofd-mannitol to d-fructose,d-arabinitol to d-xylulose, andd-sorbitol to l-sorbose by transferring the C2 hydride to the pro-S position on the nicotinamide without the use of metal cations. It is specific for the C2(R) configuration of polyols with a minimum of five carbons, and no activity is measurable with mannitol 1-phosphate, fructose 6-phosphate, or 5,6-dideoxy-d-fructose. 3B. Nidetzky, unpublished results. 3B. Nidetzky, unpublished results.How specificity for polyol substrates is achieved is not well understood. Although able to use both NADH and NADPH as cofactor, the activity with NADH is greater. Other alcohol dehydrogenases use metal ions (medium-chain dehydrogenase/reductases and some long-chain dehydrogenases) or a conserved tyrosine (SDRs) for catalysis. Neither is present in long-chain MDHs, so presumably, these alcohol dehydrogenases use a novel catalytic mechanism. Biochemical data implicate an enzyme side-chain with a pKa of 9.34 (13Slatner M. Nidetzky B. Kulbe K.D. Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (48) Google Scholar). This study was undertaken to gain an understanding of how specificity is achieved for substrate and cofactor as well as elucidate the mechanism for catalysis in the structurally uncharacterized family of long-chain MDHs. Recombinant wild-type MDH fromP. fluorescens DSM 50106 was expressed inEscherichia coli and purified as previously described (13Slatner M. Nidetzky B. Kulbe K.D. Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (48) Google Scholar). The selenomethione-substituted protein was expressed in the presence of 60 mg/liter selenomethionine accompanied by amino acids inhibiting de novo synthesis of methionine (19Doublie S. Methods Enzymol. 1997; 276: 523-530Crossref PubMed Scopus (787) Google Scholar). Purification was carried out using a protocol similar to the previously described wild-type preparation (13Slatner M. Nidetzky B. Kulbe K.D. Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (48) Google Scholar). A final gel filtration step was included to obtain highly purified protein. Gel filtration was carried out on an Äktaexplorer 100 system (Amersham Biosciences) using 140-ml Superdex 75 prep-grade material packed into a 1.6/70-cm column. Approximately 15 mg of protein in 50 mm Tris, pH 7.2, were applied to the column equilibrated with 50 mm Tris, 200 mm NaCl. The protein was eluted at a flow rate of 0.75 ml/min. Fractions containing enzyme activity were pooled and concentrated to ∼11 mg/ml. The purified protein migrated as a single band in SDS-PAGE and non-denaturing anionic PAGE. Semiquantitative densitometric analysis of the Coomassie Blue-stained gels suggested that the purity of pfMDH was 99% or better. The selenium-substituted protein retains full wild-type activity. Wild-type pfMDH was concentrated to 14 mg/ml, and the buffer changed to 10 mm Tris, 25 mm NaCl, pH 7.5. Hanging drop vapor diffusion experiments were duplicated at 277 and 293 K using both apoprotein and protein solution containing 5 mm NADH. Initial crystals of a binary complex with NADH took five months to appear at 293 K over a well containing 30% (w/v) polyethylene glycol 4000, 200 mmammonium acetate, 100 mm sodium citrate, pH 5.6. A single crystal was flash-cooled in a buffer containing 75% (v/v) well solution and 25% (v/v) ethylene glycol. Diffraction intensities to 2.5 Å were collected at Stanford Synchrotron Radiation Laboratory (SSRL) beamline 9−1 and processed using the program Denzo (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38243) Google Scholar). This indicated that the spacegroup was I222 with unit cell dimensions of a = 102 Å, b = 103 Å,c = 107 Å. A Matthews' constant (V m) of 2.58 Å3/dalton implied one monomer/asymmetric unit. Because a suitable molecular replacement model could not be identified and the original protein had degraded, selenomethione-substituted protein was used to grow additional crystals. Crystallization experiments were conducted around the previously determined conditions by mixing 1 μl of protein solution and 1 μl of well solution. Crystals of size 0.1 × 0.1 × 0.2 mm took three months to appear from a protein solution containing 14 mg/ml selenomethionine pfMDH, 25 mm NaCl, 5 mm NADH, 50 μm EDTA, 10 mm dithiothreitol, 10 mm Tris, pH 7.5, over a well of 34% (w/v) polyethylene glycol 4000, 250 mm ammonium acetate, 10 mmdithiothreitol, 100 mm sodium citrate, pH 5.0. These crystals were used for seeding, and square rods of size 0.15 × 0.15 × 0.5 mm were obtained within 2 days. Crystals were flash-cooled in a buffer containing 25% (v/v) glycerol, 75% (v/v) well solution. Lattice constants of a = 102.23 Å,b = 103.28 Å, and c = 106.57 Å were observed, similar to those found in the wild-type crystals. Several attempts to get mannitol bound in the active site were made. Ultimately, a protein solution of 14 mg/ml selenomethionine-substituted pfMDH, 25 mm NaCl, 10 mmd-mannitol, 5 mm NAD+, 50 μm EDTA, 10 mm Tris, pH 7.5, was used to grow seeded crystals over identical well conditions. Initially, these crystals were flash-cooled in a buffer containing 300 mmmannitol, 1 mm NAD+, 36% (w/v) polyethylene glycol 4000, 250 mm ammonium acetate, 100 mmsodium citrate, pH 5.0. Electron density maps calculated using data collected from these crystals suggested incomplete incorporation of mannitol. Data used for refinement of the ternary complex were obtained using crystals soaked in this buffer for 12 h before flash cooling. The binary complex multiwavelength anomalous dispersion (MAD) data set was collected at SSRL beamline 9-2. A three-wavelength MAD experiment was conducted with a high energy reference after performing a fluorescence scan to determine peak and inflection points (Table I). Diffraction intensities were processed with Denzo and Scalepack (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38243) Google Scholar). All eleven of the expected selenium sites were determined using the program Solve that resulted in a figure of merit of 0.468. After density modification executed by Resolve, the figure of merit was 0.638 and maps were calculated to 2.3-Å resolution (21Terwilliger T.C. Berendzen J. Acta Crystallogr. Sec. D. 1999; 55: 849-861Crossref PubMed Scopus (3216) Google Scholar). The density for protein and cofactor was easily interpretable, and the model was built using the program O (22Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). A data set to 1.7-Å resolution was subsequently collected on a binary crystal at SSRL beamline 9-2. The crystals were isomorphous, and the model built from the MAD data was used as starting model.Table IData collectionData setSe peakSe remoteSe inflectionHolo-nativeTernaryWavelength (Å)0.97910.93220.97930.98010.9198Resolution (Å) (highest shell)30–2.3 (2.34–2.3)30–1.7 (1.76–1.7)30–1.8 (1.84–1.8)Reflections (observed/unique)127,020/25,768126,991/25,738127,004/25,745227,038/58,258367,595/105,300Completeness (%)100 (100)100 (99.9)100 (100)94.4 (70.8)99.5 (96.1)I/ς(I)15.7 (5.1)19.0 (5.8)18.7 (5.8)15.1 (2.3)18.4 (2.8)Rmerge0.092 (0.298)0.079 (0.288)0.077 (0.296)0.083 (0.228)0.064 (0.371)Values in parenthesis are for data in highest shell. Open table in a new tab Values in parenthesis are for data in highest shell. Diffraction intensities to 1.8 Å were collected on an MDH·NAD+·mannitol complex crystal at SSRL beamline 9-1. The program Denzo was used to determine a primitive orthorhombic lattice of dimensions of a = 107.0 Å,b = 104.5 Å, and c = 101.5 Å. Systematic extinctions indicated the that the spacegroup wasP21212. The calculation of the Matthews' constant (V m) of 2.6 Å3/dalton implied two protein molecules/asymmetric unit. Molecular replacement using the holo-MDH structure stripped of water molecules as a search model was implemented using the program EPMR (23Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sec. D. 1999; 55: 484-491Crossref PubMed Scopus (688) Google Scholar). An initial solution was found for two molecules using data between 30 and 4 Å, which yielded a correlation coefficient of 0.603 and an initial R cryst = 0.41 in this resolution range. In both cases before refinement commenced, 5% of the data was flagged for calculation of R free. Alternating rounds of manual fitting and crystallographic refinement using the programs O and CNS (24Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16915) Google Scholar) resulted in the final structures of which statistics appear in Table II. Ordered water molecules were picked in CNS and manually checked for appropriate hydrogen bonding. Despite the inclusion of 1 mm NAD+ in the mannitol soak, the density of the NAD+ indicated occupancy of 0)58,319104,464Completeness (%)94.398.8Rcryst (%)17.117.6Rfree(%)19.720.2Rmsd bond lengths (Å)0.0170.016Rmsd bond angles (°)1.791.68Protein atoms38297658NADH atoms4488 (80% occupancy)Water molecules437630Mannitol atoms—24Temperature factors (Å2)Protein chain A18.5219.80Protein chain B—23.05NADH14.2328.89 (80% occupancy)Mannitol—13.48Waters31.6628.57Rmsd, root mean square deviations. Open table in a new tab Rmsd, root mean square deviations. The structure of the binary complex of pfMDH with NADH was initially determined to 2.3 Å resolution by MAD using selenomethione-substituted protein (TableI). A data set to 1.7 Å was subsequently collected and used for refinement (TableII). Crystals of a ternary complex of pfMDH with NADH and mannitol were obtained by including 10 mmd-mannitol in the protein solution and soaking the crystals for 12 h in a solution containing 300 mm mannitol. Diffraction intensities to 1.8 Å resolution were collected on a ternary crystal (Tables I and II). In both cases, the model for MDH includes residues 1–492. The final C-terminal residue was disordered and not fit. The holo model includes 1 pfMDH monomer, 1 NADH molecule, and 437 water molecules. The model of the ternary complex includes 2 pfMDH protein chains, 2 NAD+molecules with 80% occupancy, and 630 water molecules. Root mean square deviations between holo- and ternary models are <0.8 Å for α-carbons and 1.0 Å for all atoms. A Ramachandran plot as implemented by Procheck for the binary model indicates that 89.6% of the residues lie in the most favored regions, 9.9% in additional allowed regions and 0.2% in generously allowed regions. A Procheck Ramachandran plot for the ternary model indicates that 90.8% of the residues are in the most favored regions and 9.0% in additional allowed regions (25Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). In both the binary and ternary models, residue Thr-132 was the single exception and was always located in a disallowed region. This residue is located in a loop involved in cofactor binding, and density clearly indicates it is in this conformation. Consistently low temperature factors of Thr-132 and clear 2F o − F c density provide clear evidence that it is very well ordered. The structure reveals that pfMDH folds into two main domains (Fig. 1). Overall dimensions are 65 × 45 × 60 Å with the active site located at the bottom of a cleft between the two domains that is 12 × 11 Å wide and 23 Å deep. The structure confirms the biochemical data in which there are no metal ions involved in catalysis (13Slatner M. Nidetzky B. Kulbe K.D. Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (48) Google Scholar). The N-terminal domain (domain 1) includes 9 α-helices, 14 β-strands, and 3 310-helices (Fig. 2). The largest β-sheet is a six-stranded parallel dinucleotide binding motif commonly known as a Rossmann fold. A tetra-peptide linker of sequence Thr-Asp-Asp-Val connects domain 1 and the C-terminal domain (domain 2). Domain 2 contains 11 α-helices, 2 310-helices, and a small β-hairpin. The most important secondary structural elements involved in contacts between the two domains are loop regions on domain 1 and helices α10 and α15 in domain 2. In addition, helix α13 contributes a conserved arginine, Arg-361, which is engaged in interdomain salt bridges to Asp-230 on β11 and Glu-259 located on the loop at the C terminus of β12.Figure 2Schematic diagram of MDH secondary structure. β-Strands are colored blue and numbered β1–β16; strands within the canonical dinucleotide binding motif are also labeled sequentially as A–F. α-Helices arecolored green and numbered 1–20.View Large Image Figure ViewerDownload (PPT) The active site is at the interface of the two domains with the majority of residues that bind NADH contributed by the N-terminal domain and residues that bind mannitol coming primarily from the C-terminal domain. Arg-373 is the only residue from the C-terminal domain that interacts directly with the bound NAD+. Important secondary structural elements that contribute residues to the active site are α1 and loop regions at the C termini of β3, β7, and β10 from domain 1 and α10 from domain 2. A conserved lysine located on α10, Lys-295, is poised to act as the general acid/base in the reaction. Residues 1–285 form the NAD+-binding domain, domain 1. Helix α1 is in the center of domain 1 and is surrounded by the six-strand parallel β-sheet of the dinucleotide binding motif, a three-strand β-sheet formed by the extension of the final strand in the Rossmann fold with two antiparallel strands, and a four-strand mixed β-sheet. Although the relative order of the strands in the parallel sheet are the same as a Rossmann fold (CBADEF), there are insertions within it that suggest that the additional elements are not merely appended (Fig. 2). The three-strand sheet is formed wholly from residues inserted between the last strand in the dinucleotide binding fold (strand F) and the C-terminal domain, whereas the four-strand sheet is formed from residues at the N terminus, an insertion between strands B and C and the C-terminal insertion. A small solvent-accessible β-hairpin is located at the C terminus of β7. Asn-191 and Asp-230 are the only residues from domain 1 that directly bind mannitol, whereas Glu-133 has a water-mediated interaction. Eleven α-helices and a small β-hairpin make up the C-terminal domain, which can be further divided into two subdomains. Residues 289–375 compose helices α10-α14, forming domain 2A, an antiparallel three-helix bundle with two short connecting helices. Domain 2B, residues 378–493, consists of α15-α20 and the β-hairpin. Helices α15, α16, and α19 form a three-helix bundle of different structural arrangement with α15 and α16 antiparallel and α16 and α19 parallel. Helix α15 has a pronounced 40° bend because of two proline residues, Pro-383 and Pro-388, in the middle of the helix. At least one proline is found in this region in all long-chain MDH sequences examined, suggesting that the bend in α15 is a common feature for the family. Helix α15 lies antiparallel to α10 and contributes several contacts to domain 1 including three water-mediated hydrogen bonds and one salt link between Lys-384 and Asp-140. Also located on α15 is Lys-381, which hydrogen bonds to the terminal hydroxyl of mannitol away from the point of oxidation. Residues 289–313 compose α10, the first helix in the C-terminal domain that lies near the cleft between domains 1 and 2 and provides several interdomain contacts. It also contributes three residues important in substrate binding, Asn-300, His-303, and the apparent catalytic acid/base Lys-295. Arg-361 located on α13 is involved in salt links across the domain interface to Glu-259 and Asp-230, and the loop following α13 provides hydrogen bonds and van der Waals contacts to residues Leu-88, Asp-90, and Met-258 in domain 1. Arg-373 on α14 donates hydrogen bonds to hydroxyls on carbons 3 and 4 of the substrate and is the only residue from the C-terminal domain within 4 Å of the NAD+. It is located parallel but slightly offset 3.15–3.89 Å abutting the amide on the nicotinamide, contributing weak stacking or van der Waals interactions. Although there is a lack of extensive interaction between the NAD+and domain 2, a closer inspection shows that the loops from domain 1 that recognize the NAD+ provide ∼40% of the interdomain contacts. Therefore, a conformational change upon binding NAD+ is possible and is predicted from the kinetic mechanism (13Slatner M. Nidetzky B. Kulbe K.D. Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (48) Google Scholar). The NAD+is bound with the adenine anti and the nicotinamidesyn between the two domains. The sugar pucker of the adenosine ribose is C2′-endo, and the nicotinamide ribose is C3′-endo. There is an intramolecular hydrogen bond between the nicotinamide N7 and pyrophosphate oxygen NO1. Using the method of Lee and Richards (26Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5288) Google Scholar) with a probe of radius 1.4 Å, 80% of the accessible surface of NAD+ is buried in the complex with pfMDH (26Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5288) Google Scholar). Cofactor contacts are mainly from domain 1 and primarily from the canonical dinucleotide binding motif. As is commonly found in enzymes with a Rossmann fold, the pyrophosphate moiety is situated at the N terminus of α1. The glycine-rich turn in the sequence 31HIGVGGFHR39 precedes α1. HXGXGXXXR is the conserved fingerprint motif for the long chain MDH family with the exception of altronate dehydrogenases that have a glutamine at position 31. A hydrophobic Ile, Leu, or Phe follows this residue. The Nε of invariant Arg-39 orients the amide of Gly-36 to interact with the pyrophosphate by hydrogen bonding to the carbonyl oxygen of Gly-35. Phe-37 stacks against the A side of the nicotinamide, making the B side accessible to substrate and promoting transfer of the 4-pro-S hydride. A Phe or Ile is present at this position for all members of the long-chain MDH family and probably stacks against the pyridine ring. Thr-233, located on the loop following β11, has main-chain hydrogen bonds to the cofactor amide oxygen and nitrogen (Fig. 3). The adenine packs against Ile-131 and is shielded from solvent by Arg-66. Isomerization of the loop containing residues 65–69 is required for cofactor binding and release and could explain why the release of NADH is the rate-limiting step in the direction of oxidation of mannitol (13Slatner M. Nidetzky B. Kulbe K.D. Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (48) Google Scholar). Loops at the C termini of strands B, D, and E contribute residues that hydrogen bond with the ribose hydroxyls. Although significant activity with NADPH is still observed, pfMDH exhibits a strong preference for NADH over NADPH (13Slatner M. Nidetzky B. Kulbe K.D. Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (48) Google Scholar). Asp-69, which hydrogen bonds to both adenosine ribose hydroxyls, almost certainly contributes toward specificity for NAD+ over NADP+ by discouraging phosphate binding. However, it is located on a mobile loop, allowing Asp-69 to have a different conformation with NADP+ bound. Arg-66 on the same loop could make favorable contacts with a phosphate moiety and partially compensate for the negative Asp-69 interaction. Other members of this fam
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