Eur J Biochem 269, 4484–4494 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03151.x The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol Mamoru Yamanishi1, Michio Yunoki1, Takamasa Tobimatsu1, Hideaki Sato1, Junko Matsui1, Ayako Dokiya1, Yasuhiro Iuchi1, Kazunori Oe1, Kyoko Suto2, Naoki Shibata2, Yukio Morimoto2, Noritake Yasuoka2 and Tetsuo Toraya1 Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama; 2Department of Life Science, Himeji Institute of Technology, Hyogo, Japan Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity The subunit composition of the enzyme was most probably a2b2c2 When (R)- and (S)propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) ¼ 1.5) The crystal structure of glycerol dehydratase in complex with cyanoco˚ balamin and propane-1,2-diol was determined at 2.1 A resolution The enzyme exists as a dimer of the abc heterotrimer Cobalamin is bound at the interface between the a and b subunits in the so-called Ôbase-onÕ mode with 5,6dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation The active site is in a (b/a)8 barrel that was formed by a central region of the a subunit The substrate propane-1,2diol and essential cofactor K+ are bound inside the (b/a)8 barrel above the corrin ring of cobalamin K+ is heptacoordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues These structural features are quite similar to those of diol dehydratase A closer contact between the a and b subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol Adenosylcobalamin is one of the most unique compounds in nature It is a water-soluble organometallic compound possessing a Co–C r bond and serves as a cofactor for enzymatic radical reactions including carbon skeleton rearrangements, heteroatom eliminations and intramolecular amino group migrations [1] Diol dehydratase (EC 4.2.1.28) of Klebsiella oxytoca is an adenosylcobalamin (AdoCbl1) dependent enzyme that catalyzes the conversions of 1,2-diols, such as propane-1,2-diol, glycerol, and 1,2ethanediol, to the corresponding aldehydes [2,3] (Fig 1) This enzyme has been studied intensively to establish the mechanism of action of AdoCbl [4–7] The structure– function relationship of the coenzyme has also been investigated extensively with this enzyme [5–8] Recently, we have reported the three-dimensional structures of its complexes with cyanocobalamin [9] and adeninylpentylcobalamin [10] and theoretical calculations of the entire energy profile along the reaction pathway with a simplified model [11–13] In this sense, together with methylmalonyl-CoA mutase [14], glutamate mutase [15], and class II ribonucleotide reductase [16], diol dehydratase, is one of the most suitable systems with which to study the structure-based mechanisms of the AdoCbl-dependent enzymes [17,18] Glycerol dehydratase (EC 4.2.1.30) catalyzes the same reaction (Fig 1) as diol dehydratase [19–21] Although this enzyme is isofunctional with diol dehydratase, these two enzymes bear different physiological roles in the bacterial metabolisms [6,7] Selected genera of Enterobacteriaceae, such as Klebsiella and Citrobacter, produce both glycerol and diol dehydratases, but the genes for them are independently regulated [22–25]: glycerol dehydratase is induced when Klebsiella pneumoniae grows in the glycerol medium, whereas diol dehydratase is fully induced when it grows in the propane-1,2-diol-containing medium, but only slightly in the glycerol medium Glycerol dehydratase is a key enzyme for the dihydroxyacetone (DHA) pathway [23,26,27], and its genes are located in the DHA regulon [28,29] On the other hand, diol dehydratase is a key enzyme for the anaerobic degradation of 1,2-diols [30,31], and its genes are located in the pdu operon [32–34] Furthermore, although glycerol and diol enzymes are similar in their Correspondence to T Toraya, Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700–8530, Japan Fax: + 81 86 2518264, E-mail: toraya@biotech.okayama-u.ac.jp Abbreviations: AdoCbl, adenosylcobalamin; aD, bD, and cD, a, b, and c subunits of diol dehydratase; aG, bG, and cG, a, b, and c subunits of glycerol dehydratase; buffer A, 0.05 M potassium phosphate buffer (pH 8); IPTG, isopropyl thio-b-D-galactoside (Received 11 June 2002, revised 23 July 2002, accepted 25 July 2002) Keywords: coenzyme B12; adenosylcobalamin; glycerol dehydratase; crystal structure; radical enzyme catalysis Ó FEBS 2002 Structure of B12-dependent glycerol dehydratase (Eur J Biochem 269) 4485 Fig Conversion of 1,2-diols to the corresponding aldehydes by diol dehydratase subunit structures, there are several distinct differences between them in the following properties: the rate of suicide inactivation by glycerol, substrate spectrum, monovalent cation requirement, affinity for cobalamins, and immunochemical cross-reactivity [6,7] In this paper, we report the method of purifying recombinant apoglycerol dehydratase from overexpressing Escherichia coli cells and the crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane1,2-diol We intended to explain the above-mentioned differences between two dehydratases by comparing the three-dimensional structure of this enzyme with that of diol dehydratase [9,10] EXPERIMENTAL PROCEDURES Materials Crystalline AdoCbl was a gift from Eizai, Tokyo, Japan DEAE-cellulose was purchased from Wako, Osaka, Japan The other chemicals were analytical grade reagents Preparation of expression plasmids for His6-tagged glycerol dehydratase and its His6-tagged b subunit DNA segments encoding carboxyl terminal region of the glycerol dehydratase a subunit was amplified by PCR using pUSI2E(GD) [28], pfu DNA polymerase (Stratagene) and pairs of primers 5¢-TCTGAGTGCGGTGGAAGAGATG ATGAAGCG-3¢ and 5¢-AGATCTTATTCAATGGTGT CGGGCTGAACC-3¢ and digested with EcoRV and BglII Resulting 210-bp fragment was ligated with the 1.5-kb HindIII-EcoRV fragment from pUSI2E(GD) and pUSI2E digested with HindIII and BglII to yield pUSI2E(aG) DNA segments encoding the b and c subunits of glycerol dehydratase were amplified by PCR using pairs of primers 5¢-CATATGCAACAGACAACCCAAATTCAGCCC-3¢ and 5¢-AGATCTTATCACTCCCTTACTAAGTCGATG-3¢ for the b subunit and 5¢-CATATGAGCGAGAAAACCA TGCGCGTGCAG-3¢ and 5¢-AGATCTTAGCTTCCTTT ACGCAGCTTATGC-3¢ for the c subunit The segments were digested with NdeI and BglII and ligated with 3.5-kb ApaI-BglII fragment and 1.5-kb ApaI-NdeI fragment from pUSI2E(bD) [35] to yield pUSI2E(bG) and pUSI2E(cG), respectively Plasmid pUSI2E(bG) was digested with NdeI and BglII Resulting 600-bp NdeI-BglII fragment was inserted into the NdeI-BglII region of pET19b to produce pET19b(H6bG) A pair of synthetic oligonucleotides, 5¢-TATGGGCAGCAGCCATCATCATCATCATCACA GCAGCGGCCTGGTGCCGCGCGGCAGCAC-3¢ and 5¢-TAGTGCTGCCGCGCGGCACCAGGCCGCTGCT GTGATGATGATGATGATGGCTGCTGCCCA-3¢ were hybridized and inserted to the NdeI site of pUSI2E(cG) to produce pUSI2E(H6Gc) pUSI2E(bG) was digested with BamHI and BglII The resulting 0.7-kb DNA fragment was ligated with BglII-digested pUSI2E(aG) to produce pUSI2E(aGbG) The 0.5-kb BamHI–BglII fragment from pUSI2E(H6cG) was ligated with BglII-digested pUSI2E(H6cG) to produce plasmid pUSI2E(aGbGH6cG) Purification of recombinant glycerol dehydratase Glycerol dehydratase was purified from recombinant Escherichia coli by a conventional procedure (method 1) or Ni-nitrilotriacetate affinity chromatography (method 2) Substrate propane-1,2-diol was added to all the buffers used throughout the purification steps to minimize dissociation of the enzyme into components A and B [36] All operations were carried out at 0–4 °C Method Recombinant E coli JM109 harboring expression plasmid pUSI2E(GD) [28] was aerobically grown at 37 °C in Luria–Burtani (LB) medium containing propane1,2-diol (0.1%) and ampicillin (50 lgỈmL)1) to D600  0.9, induced with mM isopropyl thio-b-D-galactoside (IPTG) for h, and harvested by centrifugation Harvested cells were resuspended in buffer A (0.05 M potassium phosphate buffer; pH 8) containing mM phenylmethanesulfonyl fluoride and disrupted by sonication for 10 min, followed by centrifugation Ammonium sulfate was added to the supernatant to a final concentration of 50% saturation After 60 at °C, the suspension was centrifuged, and the precipitate was re-dissolved in buffer A The solution was subjected to gel filtration on Sepharose 6BÒ, and peak fractions containing the enzyme were pooled, dialyzed for 12 h against 40 volumes of 1.5 mM potassium phosphate buffer (pH 8) containing 0.2% propane-1,2-diol with a buffer exchange, and loaded on to a hydroxyapatite column which had previously been equilibrated with mM potassium phosphate buffer (pH 8) containing 0.2% propane1,2-diol After washing the column with mM potassium phosphate buffer (pH 8) containing 0.2% propane-1,2-diol, the enzyme was eluted with 13 mM potassium phosphate buffer (pH 8) containing 2% propane-1,2-diol The eluate was concentrated and loaded on to a Sephadex G-200Ò column which had previously been equilibrated with 20 mM potassium phosphate buffer (pH 8) containing 2% propane-1,2-diol The enzyme was eluted with the same buffer, and peak fractions containing the enzyme were pooled Method Recombinant E coli JM109 harboring pUSI2E(aGbGH6cG) was aerobically grown at 30 °C in terrific broth containing propane-1,2-diol (0.1%) and ampicillin (50 mg mL)1) to D600  0.9, induced with mM IPTG for h, and harvested by centrifugation Harvested cells were resuspended in buffer A containing mM phenylmethanesulfonyl fluoride and sonicated as described above The extract containing His6-tagged enzyme was mixed with an equal volume of buffer A containing 20 mM imidazole and 600 mM KCl and loaded on to an Ó FEBS 2002 4486 M Yamanishi et al (Eur J Biochem 269) Ni-nitrilotriacetate agarose gel (Qiagen GmbH, Germany) column which had previously been equilibrated with buffer A containing 10 mM imidazole and 300 mM KCl After washing the column with buffer A containing 10 mM imidazole and 300 mM KCl, the enzyme was eluted with buffer A containing 50–100 mM imidazole and 300 mM KCl After dialysis against 40 volumes of 50 mM TrisHCl buffer (pH 8) containing 2% propane-1, 2-diol, 150 mM KCl and 2.5 mM CaCl2, His6-tagged enzyme was digested with thrombin at 25 °C for 120 and run through the Ni-nitrilotriacetate agarose column to remove the His6-tag peptide Because a part of the enzyme had lost the b subunit, the enzyme solution was concentrated and supplied with purified b subunit by incubation at 30 °C for 30 min, followed by Sepharose 6BÒ gel filtration to remove unbound, excess b subunit The b subunit was purified from E coli BL21 (DE3) carrying pET19b(H6bG), as described above for glycerol dehydratase staining for glycerol dehydratase was performed as described previously for diol dehydratase [32] The apparent molecular weight of the enzyme was estimated by the nondenaturing PAGE on a Multigel 2–15% gradient gel (Daiichi Pure Chemicals, Tokyo, Japan) [44] Kinetic analysis of the enzyme Substrate-free enzyme used for measuring Km values for substrates was obtained by gel filtration on Sephadex G-25Ò or dialysis for days against 500 volumes of buffer A with several changes One-minute assay was employed for measurement of Km for glycerol, as glycerol induces suicide inactivation of the enzyme [39] Apparent Km values for substrates and AdoCbl were determined at an AdoCbl concentration of 15 lM and at a fixed propane-1,2-diol concentration of 100 mM, respectively EPR measurements Enzyme and protein assays Glycerol dehydratase activity was determined by a 3-methyl-2-benzothiazolinone hydrazone method [37] or an NADH–alcohol dehydrogenase coupled method [38] at 37 °C Propane-1,2-diol was used as a substrate for routine assays because glycerol acts as both a good substrate and a potent suicide inactivator [39] One unit of glycerol dehydratase is defined as the amount of enzyme activity that catalyzes the formation of lmol of propionaldehyde per minute under the assay conditions Protein concentration of crude enzyme was determined by the method of Lowry et al [40] with crystalline bovine serum albumin as a standard The concentration of purified enzyme was determined by measuring the absorbance at 280 nm The molar absorption coefficient at 280 nm calculated by the method of Gill and von Hippel [41] for this enzyme is 112 100 M)1 cm)1 The complex of glycerol dehydratase with adenosylcobinamide 3-imidazolylpropyl phosphate [45] was formed by incubating apoenzyme (100 units, 4.55 nmol) at 25 °C for with 50 nmol of the coenzyme analog in 0.65 mL of buffer A (pH 8) under a nitrogen atmosphere Propane-1,2diol (50 lmol) was then added After the mixture had been incubated at 25 °C for an additional 30 min, the mixture was quickly frozen in an isopentane bath (cooled to  )160 °C) and then in a liquid nitrogen bath The sample was transferred to an EPR cavity and kept at )130 °C with a cold nitrogen gas flow controlled by a JEOL JES-VT3A temperature controller EPR spectra were taken at )130 °C on JEOL JES-RE3X spectrometer modified with a Gunn diode X-band microwave unit under the same conditions as those described for diol dehydratase [46] Crystallization and data collection Separation of the enzyme into components A and B A purified preparation of the enzyme (80 units) was applied to a column (bed volume, 2.0 mL) of DEAE cellulose that had been equilibrated with 10 mM potassium phosphate buffer (pH 8) containing 10 mM propane-1,2-diol After washing the column with 50 mL of 10 mM potassium phosphate buffer (pH 8), components A and B were eluted successively with mL of 10 mM potassium phosphate buffer (pH 8) containing 40 mM KCl and then with 50 mL of 10 mM potassium phosphate buffer (pH 8) containing 300 mM KCl, respectively Five-milliliter fractions were collected Neither component alone was active, while the enzyme activity was restored upon addition of the other component Therefore, components A and B were assayed by adding an excessive amount of one component and making the other rate-limiting PAGE and activity staining of glycerol dehydratase PAGE was performed under nondenaturing conditions as described by Davis [42] in the presence of 0.1 M propane-1,2diol [32], or under denaturing conditions as described by Laemmli [43] Protein was stained with Coomassie brilliant blue G-250 Densitometry was carried out by Personal Scanning Imager PD110 (Molecular Dynamics) Activity Purified glycerol dehydratase (64 mgỈmL)1) in 20 mM potassium phosphate buffer (pH 8) containing 2% propane-1,2-diol was converted to the enzymcyanocobalaminỈpropane-1,2-diol complex by the same method as that for diol dehydratase [9] except that lauryldimethylamine oxide was not included The complex was crystallized by the sandwich-drop vapor diffusion method at °C X-ray diffraction data were collected at 100 K using the Quantum4R CCD detector (ADSC) on the BL40B2 beam line at SPring-8, Japan (Table 1) Reflection data were indexed, integrated and scaled using the programs Mosflm and SCALA in the CCP4 suite [47] with DPS [48] Structure determination and refinement The structure of the enzyme was determined and refined using the program CNS [49] The models were built using Xfit of XTALVIEW [50] and checked by PROCHECK No noncrystallographic symmetry (NCS) restraints were enforced during whole refinements For adjusting the positions of atoms, a composite-omit map (2Fo ) Fc) and an Fo ) Fc map were used ˚ A data set obtained was up to 2.0 A resolution Crystallographic data are listed in Table The a, b and c subunits of glycerol dehydratase show substantially high Ó FEBS 2002 Structure of B12-dependent glycerol dehydratase (Eur J Biochem 269) 4487 Table Statistics of data collection and structure determination The values in parentheses are for the highest resolution shell Data collection Refinement X-ray source Detector ˚ Wavelength (A) Temperature (K) Space group ˚ Unit cell (A) a b c b (°) ˚ Resolution (A) Measured reflections Unique reflections Completeness (%) Rmerge ˚ Resolution range (A) Used reflections Completeness (%) Rworka Rfreeb SPring-8 BL40B2 ADSC Quantum-4R 0.816 100 P21 81.4 108.2 113.1 96.8 2.0 958 394 130 635 99.6 (97.6) 0.097 (0.38) 45.0–2.1 113 453 99.9 0.208 0.248 R-factor=S||Fo| ) |Fc||/S|Fo| Rwork or the working R-factor is calculated on the 90% of the observed reflections used for the refinement b Rfree or the free R-factor is calculated on the 10% of reflections excluded from the refinement a homology to the corresponding subunits of diol dehydratase: their identities are 71, 58 and 54%, respectively, and their similarities 87, 78 and 73%, respectively [28] We started the structure determination by the molecular replacement method with the abc heterotrimer unit of diol dehydratase from Protein Data Bank (PDB) entry 1DIO [9] as a reference structure After a cross-rotation search, multiple translation searches were performed, and the monitor and the packing values were checked to determine the result from the candidates We concluded that there is an (abc)2 dimer in an asymmetric unit of the cell The ˚ calculated VM value was 3.03 A3ỈDa)1 (VM ẳ Vcell/ZặMr, where Vcell and Z are the unit cell volume and the number of protein molecules per unit cell, respectively) At this stage, the residues of diol dehydratase were replaced with the corresponding residues of glycerol dehydratase After one set of rigid-body refinement and simulated annealing were applied, a composite-omit map (2Fo ) Fc) was calculated On this map, distinct electron densities were observed in the positions next to N- and C-ends of each chain They could be assigned to certain amino-acid residues, because the C-terminal three residues of aD, the N-terminal 45 residues of bD, and the N-terminal 36 residues and the C-terminal three residues of cD were missing in the reported structure of diol dehydratase [9] In addition, aG and bG are longer by one amino acid than aD in the N-terminal and by three than bD in the C-terminal, respectively In the final structure, all residues of the a chain (Met1–Glu555), all but N-terminal 10 residues of the b chain (Phe11–Glu194), and all but N-terminal residues of the c chain (Lys4–Ser141) could be assigned to the electron density map In glycerol dehydratase, the numbers of missing residues were smaller than those of diol dehydratase We have not determined yet whether these missing residues are actually truncated by hydrolysis or not visible because of their high mobility After the successive repeats of modeling, energy-minimization and simulated annealing, about 900 water molecules were picked up, and B-factors for all the atoms were refined The structure showed good stereochemistry with root˚ mean-square (rms) deviations of 0.006 A from the ideal bond length and 1.30° from ideal bond angles The resulting Rwork and Rfree were 0.208 and 0.248, respectively, in the ˚ resolution range of 45.0–2.1 A Unless otherwise stated, structural figures were created with MOLSCRIPT [51] and RASTER3D [52] Accession number The atomic coordinates have been deposited in the Protein Data Bank with an accession code of 1IWP RESULTS AND DISCUSSION Purification and characterization of recombinant glycerol dehydratase Recombinant nontagged glycerol dehydratase was purified by a conventional method As shown in Table 2, glycerol dehydratase overexpressed in E coli was purified by ammonium sulfate fractionation and chromatography on Sepharose 6BÒ, hydroxyapatite, and Sephadex G-200Ò Table Purification of recombinant glycerol dehydratase Total activity (units) Purification step Method 1a Crude extract (NH4)2SO4 fractionation Sepharose 6BÒ Hydroxyapatite Sephadex G-200Ò Method 2b Crude extract Ni-nitrilotriacetic Thrombin digested/Ni-nitrilotriacetic Sepharose 6BÒ a Purification from 4.7 g of wet cells b Total protein (mg) Specific activity (mg)1) Yield (%) Purification (fold) 12 400 11 500 10 500 8760 7780 823 376 185 141 119 15.1 30.6 56.8 62.1 65.4 100 93 85 71 63 2.0 3.8 4.1 4.3 46 000 24 100 9770 8450 1920 234 105 70.6 24.0 100 93 120 100 52 21 18 4.2 3.9 5.0 Purification from 13 g of wet cells 4488 M Yamanishi et al (Eur J Biochem 269) (method 1) The enzyme was purified 4.3-fold in a yield of 63% Specific activity was about 65 units/mg SDS/PAGE analysis showed that three bands with an Mr of 61 000 (a), 22 000 (b) and 16 000 (c) (marked with an arrowhead) were overexpressed in E coli carrying pUSI2E(GD) (Fig 2A) and progressively enriched upon purification, and that only these subunits were found in the purified preparation of the enzyme When the enzyme was electrophoresed under Fig Characterization of purified glycerol dehydratase by PAGE SDS/PAGE (A, E) and nondenaturing PAGE (B, F) analyses of the enzyme at each purification step of method (A, 13.5% gel; B, 7.5% gel) and method (E, 12% gel; F, 7.5% gel) Resolved components A and B were also subjected to SDS/PAGE (C) and nondenaturing PAGE (D) Proteins were stained by Coomassie brilliant blue G Molecular mass markers, Sigma SDS-7 L Positions of the a, b and c subunits and their complexes are indicated with arrowheads in the right H6-a2b2c2 and H6-c represent the His6-tagged a2b2c2 complex and His6-tagged c subunit, respectively Ó FEBS 2002 nondenaturing conditions in the presence of substrate (Fig 2B), however, two bands were seen upon protein staining The ratio of the upper protein band to the lower one was estimated to be approximately by densitometric scanning Activity staining of the enzyme indicated that the upper band reconstituted catalytically active holoenzyme with added AdoCbl, but the lower one did not (data not shown) The mobility of the upper band was identical with that of active glycerol dehydratase in the extract of K pneumoniae ATCC 25955 (data not shown) Two-dimensional PAGE showed that the upper band consisted of the a, b and c subunits in a molar ratio of 1.0 : 1.0 : 1.2 The lower band was composed of the a and c subunits in a molar ratio of 1.0 : 0.9 When the purified enzyme was subjected to nondenaturing PAGE on a Multigel 2/15 gradient gel [44], two bands appeared upon protein staining, and only the upper one stained upon activity staining (data not shown) The Mr values for the upper and lower bands were 220 000 and 200 000, respectively These data suggest that the subunit compositions of the proteins in the upper and lower bands are most likely a2b2c2 (active apoenzyme, predicted molecular mass of 196 236 Da) and a2c2 (component B, predicted molecular mass of 153 526 Da), respectively In order to confirm this assignment for the lower band, we attempted to see what happens if component A is added to the purified preparation of the enzyme We prepared components A and B by separation of purified enzyme upon DEAE–cellulose chromatography in the absence of substrate Recoveries of activity of components A and B were 24% and 10%, respectively, although weak glycerol dehydratase activity was observed in the Ôcomponent BÕ fraction alone SDS/PAGE analysis showed that components A and B contain the b subunit alone and a : mixture of the a and c subunits, respectively (Fig 2C) Thus, it was concluded that the inactive protein contaminated in the purified enzyme (lower band in Fig 2B) is component B When an excessive amount of component A was added to the purified enzyme, propane-1,2-diol-dehydrating activity increased by 59% PAGE analysis under nondenaturing conditions showed that the catalytically inactive lower band seen in the purified enzyme was converted to the active upper band upon the addition of component A (Fig 2D) Three bands were observed in the Ôcomponent BÕ fraction upon nondenaturing PAGE Positions of the top and middle bands coincided well with the two bands observed with the purified enzyme Thus, it was suggested that the middle and top minor bands of the Ôcomponent BÕ fraction correspond to component B (a2c2) and a trace of contaminating active apoenzyme, a2b2c2 The bottom major band that had newly appeared has not been identified yet For brevity, we developed a quick purification method (method 2) for His6-tagged component A and glycerol dehydratase His6-tagged enzyme was overexpressed in E coli and purified with an Ni-affinity column After removal of His6 tag by digestion with thrombin, followed by passage through the Ni-nitrilotriacetate column, the enzyme obtained was incubated with excess component A and run through a Sepharose 6BÒ column Highest specific activity of the enzyme (120 mg)1) was obtained by this simple procedure (Table 2) PAGE analysis under denaturing (Fig 2E) and nondenaturing conditions (Fig 2F) indicated that the purified enzyme was contaminated with Ó FEBS 2002 Structure of B12-dependent glycerol dehydratase (Eur J Biochem 269) 4489 component B, and that the contaminating component B recombined with the b subunit (component A) to form a2b2c2 that resisted dissociation upon Sepharose 6BÒ column chromatography As a result, the enzyme was purified 5.0-fold in a yield of 18% This method was employed for crystallization of glycerol dehydratase Kinetic parameters and stereospecificity of recombinant glycerol dehydratase Kinetic constants of the purified recombinant glycerol dehydratase for AdoCbl, propane-1,2-diol, and glycerol were in reasonable agreement with those reported previously for the enzyme from K pneumoniae (Table 3), suggesting that the recombinant enzyme and the enzyme from K pneumoniae are not distinguishable When (R)- and (S)propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer (Table 3) The affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer [Km(R)/Km(S) ¼ 1.5] This preference to the (S)-isomer is significantly less marked than that reported with diol dehydratase [53,54] EPR spectroscopic evidence for the ‘base-on’ mode of cobalamin binding To identify the Co-coordinating base, EPR spectra of the suicidally inactivated complexes of the enzyme with unlabeled and [imidazole-15N2]-labeled adenosylcobinamide 3-imidazolylpropyl phosphate were compared The EPR spectra obtained with these analogs were exactly the same as those reported for diol dehydratase [46,55] (data not shown) With the unlabeled imidazolyl analog, each line of the hyperfine octet (coupling constant, 10.6 mT) showed superfine splitting into triplets (coupling constant, 1.9 mT) With the [imidazole-15N2]-labeled analog, on the other hand, the hyperfine lines (coupling constant, 10.7 mT) showed superhyperfine splitting into doublets (coupling constant, 2.7 mT) The ratio of the coupling constant with 14 N (A14N) to that with 15N (A15N) was 0.704, which is in good agreement with the theoretical one that can be calculated as follows: A14N =A15N ẳ c14N =c15N ẳ 0:713 theoreticalị where c is a gyromagnetic ratio These lines of evidence indicated that the axial ligand to Co(II) is the imidazole of the coenzyme analog Therefore, it is evident that, like diol dehydratase [46,56], glycerol dehydratase binds AdoCbl in the so-called Ôbase-onÕ mode This conclusion is consistent with the finding of Poppe et al that p-cresolylcobamide coenzyme is inactive and serves as an inhibitor for diol and glycerol dehydratases [57] Overall structure of glycerol dehydratase in complex with cobalamin and propane-1,2-diol The crystal structure of glycerol dehydratase in complex with cobalamin and propane-1,2-diol was determined at ˚ 2.1 A resolution by the molecular replacement method The schematic view of the overall structure is shown in Fig 3A The enzyme exists as a dimer of the abc heterotrimer There is a noncrystallographic twofold axis around the center of Fig 3A The structure of an abc heterotrimer unit is shown in Fig 3B The central region of the a subunit constitutes the (b/a)8 barrel, the so-called TIM (triosephosphate isomerase) barrel Propane-1,2-diol, a substrate, and K+, an essential cofactor, are bound inside the barrel The active site-cavity is covered by the corrin ring of cobalamin that is bound on the interface of the a and b subunits Two a subunits form dimer a2 to which two b and two c subunits are bound separately This structure is quite similar to that of diol dehydratase [9] To compare the Ca trace between glycerol and diol dehydratases, the abc structure of glycerol dehydratase superimposed on the structure of diol dehydratase is shown in Fig 3C with the rms deviation ranges differently colored It is clear that deviations of atoms in the b and c subunits are relatively large, although the rms ˚ deviation of Ca atoms in the a subunit was less than 1.0 A The Km values of glycerol dehydratase for AdoCbl is 40–100 times lower than that of diol dehydratase (Table 3) Such higher affinity of glycerol dehydratase for AdoCbl may be explained by the closer contact between the a and b subunits in which cobalamin sits Glycerol dehydratase is isofunctional with diol dehydratase, and its amino acid sequences of the a, b and c subunits are 71, 58 and 54% identical with those of diol dehydratase [28] They are immunologically different or only slightly cross-reactive under nondenaturing conditions [22], but anti-(K oxytoca diol dehydratase) antiserum cross-reacted with K pneumoniae glycerol dehydratase to some extent under denaturing conditions (data not shown) As shown in Fig 3D, most of the amino acid residues that are not conserved between these enzymes are located on the surface of the glycerol dehydratase molecule, whereas the conserved residues constitute the core part of the enzyme This fact explains the above-mentioned very low cross-reactivity of glycerol dehydratase with anti-(diol dehydratase) antiserum under nondenaturing conditions and its low but distinct cross-reactivity under denaturing conditions Table Kinetic constants for the coenzyme and substrates Km (kcat) [mM (s)1)] Glycerol dehydratase Recombinant (method 1) Recombinant (method 2) Klebsiella pneumoniae enzyme a From [39] b Km for AdoCbl (nM) 20a From [61] c From [62] d Propane-1,2-diol 1.2 1.1 (860) 8 Glycerol 0.32 0.32 ± 0.08d (392) 1.50b 0.36c Mean ± SD, n ¼ 11–14 (R)-Propane-1,2-diol (S)-Propane-1,2-diol 0.40 ± 0.09 (604 ± 32)d 0.27 ± 0.10 (244 ± 23)d 4490 M Yamanishi et al (Eur J Biochem 269) Ó FEBS 2002 Fig Structure of glycerol dehydratase in complex with cobalamin and propane-1,2-diol (A) Overall structure One abc heterotrimer unit of the two was drawn in a schematic model, and the other in a tube model a, b and c chains are colored in blue, yellow and orange, respectively, darkening continuously from the N- to C-terminal sides Eight b strands constituting the (b/a)8 barrel are drawn in cartoon Cobalamin, propane-1,2-diol and K+ are shown as CPK models colored in pink, green, and cyan, respectively (B) Structure of the abc heterotrimer unit (C) Stereoview of the Ca ˚ traces of the abc heterotrimer unit The corresponding traces of diol dehydratase are drawn in gray Root mean square deviation (A) from the diol dehydratase structure: dark blue, < 0.5; light blue, 0.5–1.0; yellow, 1.0–1.5; orange, 1.5–2.0; red, 2.0–5; pink, > Cobalamin, propane-1,2-diol and K+ are shown as ball-and-stick models (D) Stereo drawing of the distribution of the conserved and different residues Identical and different residues are shown in a blue ball-and-stick model and colored CPK models, respectively Red, different; orange, weakly conserved; yellow, strongly conserved [63] Cobalamin-binding site and the conformation of bound cobalamin Figure 4A depicts the structure of the active site in the (b/a)8 barrel Substrate propane-1,2-diol and K+ are locked in the active-site cavity that is isolated from a bulk of water by the corrin ring of cobalamin Figure 4B shows the structure around the enzyme-bound cobalamin The cobalamin molecule is bound between the a and b subunits in the so-called Ôbase-onÕ mode ) that is, with the 5,6-dimethylbenzimidazole moiety coordinating to the cobalt atom Again, this binding mode is quite similar to that of diol dehydratase [9] Crystallographic indication of the base-on mode of cobalamin binding in class II ribonucleotide reductase of Lactobacillus leichmannii has also been reported very recently by Drennan and coworkers [16] The amino acid residues of diol dehydratase that are hydrogenbonded to the peripheral amide side chains of the corrin ring [9] are all conserved in glycerol dehydratase as well In addition to these conserved residues, the hydroxyl group of Serb122 is hydrogen-bonded to the amide oxygen of the g-acetamide side chain of the corrin ring in glycerol dehydratase (red dotted line in Fig 4B) In diol dehydratase, the corresponding residue is Prob155 that cannot form the hydrogen bond Furthermore, the lengths of the hydrogen bonds are shorter with five amino acid residues and longer with four residues in glycerol dehydratase than those in the diol enzyme These may be reminiscent of the fact that the former enzyme binds AdoCbl much more tightly than the latter enzyme (Table 3) In the case of glycerol dehydratase, no electron density of the cyanide ligand was seen even though diffraction data were collected at 100 K The Co–N bond distance between the cobalt atom and N(3) of 5,6-dimethylbenzimidazole in ˚ the glycerol dehydratase-bound cobalamin is 2.48 A This value is close to that in the diol dehydratascobalamin Ĩ FEBS 2002 Structure of B12-dependent glycerol dehydratase (Eur J Biochem 269) 4491 Fig Structures of the active site and the cobalamin-binding site (A) Stereo drawing of the active-site cavity viewed from the direction parallel to the plane of the corrin ring Activesite residues interacting with the substrate (green) and K+ (cyan) are shown in ball-andstick models Cobalamin, pink (B) Residues hydrogen-bonded to cobalamin The residues interacting with cobalamin from distances ˚ ˚ shorter by 0.1 A and longer by 0.1 A than those in diol dehydratase are colored in yellow and green, respectively The label ÔBÕ after the residue number refers to residues of the b subunit The red hydrogen bond is present only glycerol dehydratase (C) Orientation of the a-acetamide side chain of pyrrole ring A of the corrin ring A possible hydrogen bond between the a-acetamide side chain and an amino acid residue is also shown (a) Glycerol dehydratascobalamin complex (b) Diol dehydratascobalamin complex whose structure was determined at °C (PDB: 1DIO) [9] (c) Diol dehydratascyanocobalamin complex (PDB: 1EGM) [10] (d) Diol dehydratasadeninylpentylcobalamin complex (PDB: 1EEX) [10] ˚ complex (2.50 A) whose structure was determined at °C [9] and significantly longer than those in the complexes of ˚ diol dehydratase with cyanocobalamin (2.18 A) and with ˚ adeninylpentylcobalamin (2.22 A) [10] We assigned the former as the diol dehydratascob(II)alamin complex, because no electron density corresponding to the cyano group was observed [10] It has been reported that the Co–CN bond is cleaved by X-ray irradiation during data collection with diol dehydratase [58] Kratky and coworkers have reported that free and glutamate mutase-bound cyanocobalamin is reduced to cob(II)alamin by X-ray irradiation [59] Therefore, we believe that the structure reported in this paper is also that of the glycerol dehydratascob(II)alamin complex The dihedral angle of the northern and southern least-squares planes is 5.5°, indicating that the corrin ring of the glycerol dehydratasebound cobalamin is also almost planar, as compared with that of free cyanocobalamin (14.1°) This value is close to those in the diol dehydratase-bound cobalamins (2.9–5.1°) [9,10] Figure 4C indicates the comparison of the position of the a-acetamide side chain of pyrrole ring A of the corrin ring in the glycerol dehydratase-bound cobalamin (Fig 4Ca) with those in the diol dehydratase-bound cobalamins It is clear that the direction of the a-acetamide side chain is very close to that in the structure of the diol dehydratascobalamin complex determined at °C [9] (Fig 4Cb) It seems that this side chain turns to the opposite direction to the cobalt atom, depending upon the steric bulk of the upper axial ligand (CN– or adeninylpentyl group) (Fig 4Cc,d) Thus, this offers evidence that the glycerol dehydratase-bound cobalamin exists in a five-coordinated, square-pyramidal complex, suggesting again that the bound cobalamin is actually cob(II)alamin Substrate- and K+-binding sites Substrate propane-1,2-diol and the essential cofactor K+ are bound inside the TIM barrel of the a subunit (Fig 4A) This suggests that K+ bound in the active site of glycerol dehydratase in the presence of substrate is also not exchangeable with NH4+ in the crystallization solution, as in diol dehydratase [9] The two hydroxyl groups of substrate directly coordinate to K+ (Fig 5A) The O(2) and O(1) atoms of the substrate are fixed in the active site by hydrogen bonding with Glua171 and Glna297 and Hisa144 Ó FEBS 2002 4492 M Yamanishi et al (Eur J Biochem 269) Fig Interaction of glycerol dehydratase with the substrate (A) Stereo drawing of a part of the electron-density map (omit map, 2Fo ) Fc) contoured for the substrate and nearby amino acid residues K+ and propane-1,2-diol are shown as a cyan CPK model and a green stick model, respectively (B) Stereo drawing of the substrate and nearby residues Colored stick model, glycerol dehydratase; gray stick model, diol dehydratase and Aspa336, respectively (Fig 4A) K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms of the active-site residues Such characteristics of the substrate- and K+-binding sites are quite similar to those seen in diol dehydratase [9] Although racemic propane-1,2diol was used for purification and crystallization, the (R)-enantiomer is better fitted to the electron-density map (Fig 5A) When R-values were compared with (R)- and (S)-isomers in the active site, the (R)-isomer gave slightly lower values Furthermore, when Fo ) Fc maps were compared, there was no significant electron density left for the (R)-isomer, while slight electron density remained for the (S)-isomer Thus, we assigned the (R)-isomer to the electrondensity map The kinetic results, however, indicate that glycerol dehydratase shows almost equal affinity toward the (S)- and (R)-isomers (Table 3) The reason for this discrepancy is at present not clear In contrast, diol dehydratase prefers the (S)-isomer (Km(R)/Km(S) ¼ 3.1–3.2) [9] The subtle differences between glycerol and diol dehydratases in the positions of Vala301, Sera302, and Aspa336 (Fig 5B) might explain the less marked preference of glycerol dehydratase to the (S)-enantiomer in the substrate binding Glycerol serves as a very good substrate as well as a potent suicide inactivator for both glycerol dehydratase [39] and diol dehydratase [3] It is well known that diol dehydratase undergoes the inactivation by glycerol at a faster rate than glycerol dehydratase [39,60] It was reported by Bachovchin et al that diol dehydratase distinguishes between ÔRÕ and ÔSÕ binding conformations, the enzym(R)-glycerol complex being predominantly responsible for the product-forming reaction, while the enzym(S)-glycerol complex results primarily in the inactivation reaction [60] Therefore, the less marked preference of the glycerol dehydratase toward the (S)-isomer explains why it is inactivated by glycerol during catalysis at a slower rate than the diol dehydratase ACKNOWLEDGMENTS We would like to thank Dr Keiko Miura for her kind help in data collection at the BL40B2 beamline, SPring-8, Japan We thank Ms Yukiko Kurimoto for her assistance in manuscript preparation REFERENCES Banerjee, R., ed (1999) Chemistry and Biochemistry of B12 John Willy & Sons, New York Lee, H.A Jr & Abeles, R.H (1963) Purification and properties of dioldehydrase, an enzyme requiring a cobamide coenzyme J Biol Chem 238, 2367–2373 Toraya, T., Shirakashi, T., Kosuga, T & Fukui, S (1976) Coenzyme B12-dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator Biochem Biophys Res Commun 69, 475–480 Abeles, R.H (1979) Current status of the mechanism of action of B12-coenzyme In Vitamin B12 (Zagalak, B & Friedrich, W., eds), pp 373–388 Walter de Gruyter, Berlin Toraya, T & Fukui, S (1982) Diol dehydrase In B12 (Dolphin, D., ed.), Vol 2, pp 233–262 John Wiley & Sons, New York Toraya, T (1994) Diol dehydrase and glycerol dehydrase, coenzyme B12-dependent isozymes Metal Ions Biol Sys 30, 217–254 Toraya, T (1999) Diol dehydratase and glycerol dehydratase In Chemistry and Biochemistry of B12 (Banerjee, R., ed.), pp 783–809 John Wiley & Sons, New York Toraya, T (1998) Recent structure-function studies of B12 coenzymes in diol dehydrase In Vitamin B12 and B12-Proteins (Krautler, B., Arigoni, D & Golding, B.T., eds), pp 303320 ă Wiley-VCH, Weinheim Shibata, N., Masuda, J., Tobimatsu, T., Toraya, T., Suto, K., Morimoto, Y & Yasuoka, N (1999) A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase Structure 7, 997–1008 10 Masuda, J., Shibata, N., Morimoto, Y., Toraya, T & Yasuoka, N (2000) How a protein generates a catalytic radical from Ó FEBS 2002 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Structure of B12-dependent glycerol dehydratase (Eur J Biochem 269) 4493 coenzyme B12: X-ray structure of diol dehydratase-adeninylpentylcobalamin complex Structure 8, 775–788 Toraya, T., Yoshizawa, K., Eda, M & Yamabe, T (1999) Direct participation of potassium ion in the catalysis of coenzyme B12dependent diol dehydratase J Biochem 126, 650–654 Toraya, T., Eda, M., Kamachi, T & Yoshizawa, K (2001) Energetic feasibility of hydrogen abstraction and recombination in coenzyme B12-dependent diol dehydratase reaction J Biochem 130, 865–872 Eda, M., Kamachi, T., Yoshizawa, K & Toraya, T (2002) Theoretical study on the mechanism of catalysis of coenzyme B12dependent diol dehydratase Bull Chem Soc Jpn 75, 1469–1481 Mancia, F., Keep, N.H., Nakagawa, A., Leadlay, P.F., Mcsweeney, S., Rasmussen, B., Bosecke, P., Diat, O & Evans, P.R (1996) ă How coenzyme B12 radicals are generated: the crystal structure of ˚ methylmalonyl-coenzyme A mutase at A resolution Structure 4, 339–350 Reitzer, R., Gruber, K., Jogl, G., Wagner, U.G., Bothe, H., Buckel, W & Kratky, C (1999) Glutamate mutase from Clostridium cochlearium: the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights Structure 7, 891–902 Sintchak, M.D., Arjara, G., Kellogg, B.A., Stubbe, J & Drennan, C (2002) The crystal structure of class II ribonucleotide reductase reveals how an allosterically regulated monomer mimics a dimer Nat Struct Biol 9, 293–300 Toraya, T (2000) Radical catalysis of B12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases Cell Mol Life Sci 57, 106–127 Toraya, T (2002) Enzymatic radical catalysis: coenzyme B12dependent diol dehydratase Chem Rec in press Pawelkiewicz, J & Zagalak, B (1965) Enzymatic conversion of glycerol into b-hydroxypropionaldehyde in a cell-free extract from Aerobacter aerogenes Acta Biochim Polon 12, 207–218 Schneider, Z., Larsen, E.G., Jacobson, G., Johnson, B.C & Pawelkiewicz, J (1970) Purification and properties of glycerol dehydrase J Biol Chem 245, 3388–3396 Stroinski, A., Pawelkiewicz, J & Johnson, B.C (1974) Allosteric interactions in glycerol dehydratase Purification of enzyme and effects of positive and negative cooperativity for glycerol Arch Biochem Biophys 162, 321–330 Toraya, T & Fukui, S (1977) Immunochemical evidence for the difference between coenzyme-B12-dependent diol dehydratase and glycerol dehydratase Eur J Biochem 76, 285–289 Toraya, T., Kuno, S & Fukui, S (1980) Distribution of coenzyme B12-dependent diol dehydratase and glycerol dehydratase in selected genera of Enterobacteriaceae and Propionibacteriaceae J Bacteriol 141, 1439–1442 Forage, R.G & Foster, M.A (1979) Resolution of the coenzyme B-12-dependent dehydratases of Klebsiella sp & Citrobacter freundii Biochim Biophys Acta 569, 249–258 Toraya, T., Honda, S., Kuno, S & Fukui, S (1978) Coenzyme B12-dependent diol dehydratase: regulation of apoenzyme synthesis in Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 J Bacteriol 135, 726–729 Forage, R.G & Foster, M.A (1982) Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B12-dependent glycerol and diol dehydratases J Bacteriol 149, 413–419 Forage, R.G & Lin, E.C (1982) dha system mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418 J Bacteriol 151, 591–599 Tobimatsu, T., Azuma, M., Matsubara, H., Takatori, H., Niida, T., Nishimoto, K., Satoh, H., Hayashi, R & Toraya, T (1996) Cloning, sequencing and high-level expression of the genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae J Biol Chem 271, 22352–22357 Seyfried, M., Daniel, R & Gottschalk, G (1996) Cloning, sequencing, and overexpression of the genes encoding coenzyme 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 B12-dependent glycerol dehydratase of Citrobacter freundii J Bacteriol 178, 5793–5796 Hosoi, N., Morimoto, K., Ozaki, C., Kitamoto, Y & Ichikawa, Y (1978) Enzyme activities involved in the metabolism of 1,2propanediol by Propionibacterium freudenreichii J Ferment Techn 56, 566–572 Toraya, T., Honda, S & Fukui, S (1979) Fermentation of 1,2-propanediol and 1,2-ethanediol by some genera of Enterobacteriaceae, involving coenzyme B12-dependent diol dehydratase J Bacteriol 139, 39–47 Tobimatsu, T., Hara, T., Sakaguchi, M., Kishimoto, Y., Wada, Y., Isoda, M., Sakai, T & Toraya, T (1995) Molecular cloning, sequencing and expression of the genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca J Biol Chem 270, 7142–7148 Bobik, T.A., Xu, Y., Jeter, R.M., Otto, K.E & Roth, J.R (1997) Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase J Bacteriol 179, 6633–6639 Tobimatsu, T., Azuma, M., Hayashi, S., Nishimoto, K & Toraya, T (1998) Molecular cloning, sequencing and characterization of the genes for adenosylcobalamin-dependent diol dehydratase of Klebsiella pneumoniae Biosci Biotechn Biochem 62, 1774–1777 Tobimatsu, T., Sakai, T., Hashida, Y., Mizoguchi, N., Miyoshi, S & Toraya, T (1997) Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca Arch Biochem Biphys 347, 132– 140 Schneider, Z., Pech, K & Pawelkiewicz, J (1966) Enzymic transformation of glycerol to beta-hydroxypropionic aldehyde II Dissociation of the enzyme into two protein fragments Bull Acad Pol Sci 14, 7–12 Toraya, T., Ushio, K., Fukui, S & Hogenkamp, H.P.C (1977) Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme J Biol Chem 252, 963–970 Toraya, T., Krodel, E., Mildvan, A.S & Abels, R.H (1979) Role of peripheral side chains of vitamin B12 coenzymes in the reaction catalyzed by dioldehydrase Biochemistry 18, 417–426 Poznanskaya, A.A., Yakusheva, M.I & Yakovlev, V.A (1977) Study of the mechanism of action of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes II The inactivation kinetics of glycerol dehydratase complexes with adenosylcobalamin and its analogs Biochim Biophys Acta 484, 236–243 Lowry, O.H., Rosebrough, N.J., Farr, A.L & Randall, R.J (1951) Protein measurement with the folin phenol reagent J Biol Chem 193, 265–275 Gill, S.C & Von Hippel, P.H (1989) Calculation of protein extinction coefficients from amino acid sequence data Anal Biochem 182, 319–326 Davis, B.J (1964) Disc electrophoresis-II: method and application to human serum proteins Ann N Y Acad Sci 121, 404–427 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 Lambin, P & Fine, J.M (1979) Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents Anal Biochem 98, 160–168 Toraya, T & Ishida, A (1991) Roles of the D-ribose and 5,6dimethylbenzimidazole moieties of the nucleotide loop of adenosylcobalamin in manifestation of coenzymic function in the diol dehydrase reaction J Biol Chem 266, 5430–5437 Yamanishi, M., Yamada, S., Muguruma, H., Murakani, Y., Tobimatsu, T., Ishida, A., Yamauchi, J & Toraya, T (1998) Evidence for axial coordination of 5,6-dimethylbenzimidazole to 4494 M Yamanishi et al (Eur J Biochem 269) 47 48 49 50 51 52 53 54 55 the cobalt atom of adenosylcobalamin bound to diol dehydratase Biochemistry 37, 4799–4803 Collaborative Computational Project Number (1994) The CCP4 suite: programs for protein crystallography Acta Cryst D50, 760–763 Rossmann, M.G & van Beek, C.G (1999) Data processing Acta Cryst D55, 1631–1653 Brger, 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 (1998) Crystallography and NMR system: a new software suite for macromolecular structure determination Acta Cryst D54, 905–921 Mcree, D.E (1992) A visual protein crystallographic software system for X11/XView J Mol Graphics 10, 44–46 Kraulis, J (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures J Appl Crystallogr 24, 946 Merritt, E.A & Murphy, M.E.P (1994) Raster 3D, version 2.0: a program for photorealistic molecular graphics Acta Cryst D50, 869–873 Zagalak, B., Frey, P.A., Karabatsos, G.L & Abeles, R.H (1966) The stereochemistry of the conversion of D and L 1,2-propanediols to propionaldehyde J Biol Chem 241, 3028–3035 Yamane, T., Kato, T., Shimizu, S & Fukui, S (1966) Comparison of reactivity between D-propanediol and L-propanediol in intramolecular oxidation–reduction catalyzed by dioldehydrase requiring cobamide coenzyme Arch Biochem Biophys 113, 362– 366 Yamanishi, M., Yamada, S., Ishida, A., Yamauchi, J & Toraya, T (1998) EPR spectroscopic evidence for the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydratase by coenzyme analogs J Biochem 124, 598–601 Ó FEBS 2002 ´ 56 Abend, A., Nitsche, R., Bandarian, V., Stupperich, E & Retey, J (1998) Diol dehydratase binds coenzyme B12 in the Ôbase-onÕ mode ESR investigations on cob(II)alamin Angew Chem Int Ed Engl 37, 625–627 ´ 57 Poppe, L., Stupperich, E., Hull, W.E., Buckel, T & Retey, J (1997) A base-off analogue of coenzyme-B12 with a modified nucleotide loop–1H-NMR structure analysis and kinetic studies with (R)-methylmalonyl-CoA mutase, glycerol dehydratase, and diol dehydratase Eur J Biochem 250, 303–307 58 Masuda, J., Shibata, N., Morimoto, Y., Toraya, T & Yasuoka, N (2001) Radical production simulated by photoirradiation of the diol dehydratase–adeninylpenthylcobalamin complex J Synchrotron Rad 8, 1182–1185 59 Champloy, F., Gruber, K., Jogl, G & Kratky, C (2000) XAS spectroscopy reveals X-ray-induced photoreduction of free and protein-bound B12 cofactors J Synchrotron Rad 7, 267–273 60 Bachovchin, W.W., Eagar, R.G Jr, Moore, K.W & Richards, J.H (1977) Mechanism of action of adenosylcobalamin: glycerol and other substrate analogues as substrates and inactivators for propanediol dehydratase – kinetics, stereospecificity, and mechanism Biochemistry 16, 1082–1092 61 Yakusheva, M.I., Malahov, A.A., Poznanskya, A.A & Yakovlev, V.A (1974) Determination of glycerol dehydratase activity by the coupled enzymic method Anal Biochem 60, 293–301 62 Honda, S., Toraya, T & Fukui, S (1980) In situ reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase J Bacteriol 143, 1458–1465 63 Thompson, J.D., Higgins, D.G & Gibson, T.J (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673– 4680  ... and serves as an inhibitor for diol and glycerol dehydratases [57] Overall structure of glycerol dehydratase in complex with cobalamin and propane-1,2-diol The crystal structure of glycerol dehydratase. .. dehydratase- bound cobalamins (2.9–5.1°) [9,10] Figure 4C indicates the comparison of the position of the a-acetamide side chain of pyrrole ring A of the corrin ring in the glycerol dehydratase- bound cobalamin. .. dehydratascobalamin Ĩ FEBS 2002 Structure of B12-dependent glycerol dehydratase (Eur J Biochem 269) 4491 Fig Structures of the active site and the cobalamin- binding site (A) Stereo drawing of the active-site