Roles of adenine anchoring and ion pairing at the coenzyme B 12 -binding site in diol dehydratase catalysis Ken-ichi Ogura, Shin-ichi Kunita, Koichi Mori, Takamasa Tobimatsu and Tetsuo Toraya Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Japan Adenosylcobalamin (AdoCbl) is a cofactor for enzy- matic radical reactions, including carbon skeleton rearrangements, heteroatom eliminations, and intramo- lecular amino group migrations [1–3]. These reactions involve the migration of a hydrogen atom from one carbon atom of the substrate to the adjacent carbon atom [4,5] in exchange for group X, which moves in the opposite direction [6]. The reactions are initiated by abstraction of a hydrogen atom from substrates with an adenosyl radical that is generated in the active site through homolysis of the cobalt–carbon (Co–C) bond of AdoCbl [1–3,7,8]. The activation and homolysis of the Co–C bond upon coenzyme binding to apoenzyme is therefore considered to be a key step for all the AdoCbl- dependent reactions. Diol dehydratase (dl-1,2-pro- panediol hydrolyase; EC 4.2.1.28) is an enzyme that catalyzes the AdoCbl-dependent conversion of 1,2-diols and glycerol to the corresponding aldehydes [9,10]. Keywords adenine anchoring; adenosylcobalamin; coenzyme B 12 ; diol dehydratase; ion pairing Correspondence T. Toraya, Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Okayama 700-8530, Japan Fax: +81 86 251 8264 Tel: +81 86 251 8194 E-mail: toraya@cc.okayama-u.ac.jp (Received 4 September 2008, revised 8 October 2008, accepted 15 October 2008) doi:10.1111/j.1742-4658.2008.06745.x The X-ray structure of the diol dehydratase–adeninylpentylcobalamin com- plex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Sera224, and of 6-NH 2 , N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the e-NH 2 group of Lysb135 and the phosphate group of cobala- min. To assess the importance of adenine anchoring and ion pairing, Sera224 and Lysb135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sa224A, Sa224N and Kb135E mutants were 19–2% as active as the wild-type enzyme, whereas the Kb135A, Kb135Q and Kb135R mutants retained 58–76% of the wild-type activity. The presence of a positive charge at the b135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme–cobalamin interaction. The Sa224A and Sa224N mutants showed a k cat ⁄ k inact value that was less than 2% that of the wild-type, whereas for Lysb135 mutants this value was in the range 25–75%, except for the Kb135E mutant (7%). Unlike the wild-type holoenzyme, the Sa224N and Sa224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Sera224 is important for cobalt–carbon bond activa- tion and for preventing the enzyme from being inactivated. Upon inactiva- tion of the Sa224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disap- peared in EPR. 5¢-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one. Abbreviations AdePeCbl, adeninylpentylcobalamin; AdoCbl, adenosylcobalamin or coenzyme B 12 ; aqCbl, aquacobalamin; CN-Cbl, cyanocobalamin; OH-Cbl, hydroxocobalamin. 6204 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS Structure–function studies using adenine-modified analogs of AdoCbl have shown relatively low specific- ity of diol dehydratase for the adenine moiety in the adenosyl group, an upper axial ligand [11–13]. 1-Deaza and 3-deaza analogs of AdoCbl are partially active (56% and 46%, respectively) as coenzyme, whereas 7-deaza and N 6 ,N 6 -dimethyl derivatives do not show detectable coenzyme activity and act as strong compet- itive inhibitors of AdoCbl. Guanosylcobalamin is an inactive coenzyme with low affinity for the enzyme. The nucleotide loop moiety of AdoCbl is not directly involved in the catalytic process, but it is obligatory for the continuous progress of catalytic cycles [14–16]. Adenosylcobinamide methyl phosphate, an analog of AdoCbl lacking the nucleotide loop moiety, does not show detectable coenzyme activity, but behaves as a strong competitive inhibitor of AdoCbl [17]. Upon incubation with apoenzyme in the presence of sub- strate, this analog undergoes irreversible cleavage of its Co–C bond, forming an enzyme-bound Co(II)-contain- ing species. Adenosylcobinamide neither functions as a coenzyme nor binds tightly to apoenzyme. It is thus evident that the phosphate group of the coenzyme nucleotide loop is essential for tight binding to the apoenzyme and therefore for subsequent activation of the Co–C bond and catalysis. The X-ray structure of diol dehydratase showed that the enzyme exists as a dimer of heterotrimers and binds cobalamin in the ‘base-on’ mode, namely with a 5,6-dimethylbenzymidazole moiety coordinating to the cobalt atom [18], as suggested by EPR studies [19,20]. The structure of the enzyme in complex with ade- ninylpentylcobalamin (AdePeCbl), an inactive coen- zyme analog, revealed the presence of the adenine- binding pocket in the active site of the enzyme [21] (Fig. 1A). The adenine ring of the bound AdePeCbl is nearly parallel to the corrin ring and faces pyrrole ring C. It is trapped in the pocket by several hydrogen bonds with amino acid residues in the a-subunit (Fig. 1B). The overall structure of the complex is essentially the same as that of the enzyme–cyanocobal- amin (CN-Cbl) complex, except that the orientation of the side chain OH group of Sera224 is largely rotated to form a hydrogen bond with N3 of the adenine moi- ety in the enzyme–AdePeCbl complex. Sera224 is the only residue whose side chain is hydrogen-bonded with the adenine ring. This residue is conserved between diol dehydratases [22–24] and glycerol dehydratases [25–27]. Other residues also form hydrogen bonds with 6-NH 2 , N1, or N7, but through the main chain amide groups [21]. In addition, the e-NH 2 group of Lysb135 forms a salt bridge with the phosphate group of cobal- amin (Fig. 1C) [18]. In this article, we report the roles of adenine anchor- ing and ion pairing at the AdoCbl-binding site in diol dehydratase catalysis. To study the functions of Sera224 and Lysb135 by site-directed mutagenesis, we prepared several mutant enzymes, in which either Sera224 or Lysb135 is mutated to other amino acids, and investigated their catalytic properties by kinetic and spectroscopic analyses. The mechanism-based inactivation of a mutant enzyme during catalysis is also reported here. Results Expression and purification of mutant diol dehydratases Mutant apoenzymes in which Sera224 or Lysb135 was mutated to another amino acid were expressed in Esc- herichia coli cells and purified to homogeneity by the same procedure as that described for the wild-type enzyme [28] – that is, by extraction from crude mem- brane fractions with a buffer containing 1% Brij35. Purified preparations of the mutant enzymes were ana- lyzed by PAGE under denaturing and nondenaturing conditions. Upon SDS ⁄ PAGE (Fig. 2A), three bands corresponding to the a-, b- and c-subunits were observed in each mutant enzyme. Upon nondenaturing PAGE (Fig. 2B), all the mutants were electrophoresed as a single band that corresponds to the (abc) 2 complex. Catalytic activity and kinetic properties of Sera224 mutant diol dehydratases As shown in Table 1, k cat values of the Sa224A and Sa224N mutants were 19% and 5%, respectively, of that of the wild-type enzyme, but rapid inactivation took place with the Sa 224A mutant (Fig. 3A). The k cat ⁄ k inact values, which show the average numbers of catalytic turnovers before inactivation [29], indicated that both Sera224 mutants were inactivated after 8000–15 000 turnovers on average, whereas the wild- type enzyme underwent inactivation after 7.5 · 10 5 turnovers. The result obtained with the Sa224A mutant suggests that the hydrogen bond donation from the side chain OH group of Sera224 to N3 of the adenine ring of AdoCbl is important for the continu- ous progress of catalytic cycles. In the case of the Sa224N mutant, the hydrogen bond might not be formed, because the side chain –CH 2 CONH 2 group of Asn in the Sa224N mutant is longer than the –CH 2 OH group of Ser in the wild-type enzyme. To examine the possibility that such differences might affect the affinities of the enzyme for ligands, K i. Ogura et al. Residues involved in coenzyme B 12 binding FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6205 apparent K m values for the coenzyme AdoCbl and a substrate 1,2-propanediol, as well as K i values for an inhibitor, CN-Cbl, were determined with the mutants. K m values for AdoCbl (Table 2) and 1,2-propanediol (Table 1) increased markedly for the Sa224N mutant, as compared with the wild-type enzyme, whereas K i for CN-Cbl was not so much affected. In contrast, K m for AdoCbl and K i for CN-Cbl decreased slightly for the Sa224A mutant, although K m for 1,2-propanediol did not change. Thus, it became clear that the steric crowding or inappropriate hydrogen bonding induced by the relatively bulky side chain of Asn in the Sa224N mutant lowers significantly the affinity for the coenzyme and substrate as well as catalytic effi- ciency (k cat ⁄ K m ). Catalytic activities and kinetic properties of Lysb135 mutant diol dehydratases Table 1 indicates that k cat values of the Kb135R, Kb135A and Kb135Q mutants were 58–76% that of the wild-type enzyme at saturating concentrations of KK PDO PDO S224 S224 K135 K135 A BC Fig. 1. The structure of the coenzyme-binding site in diol dehydratase. (A) Stereo drawing of the hydrogen bonding and the ion pairing interactions of AdePeCbl with Sera224 (S224) and Lysb135 (K135), respectively. PDO represents (S)-1,2-propanediol in this drawing. Pink and green colors indicate the a- and b-subunits, respectively, darkening continuously from the N-terminal to the C-terminal sides. (B) Resi- dues interacting with the adenine moiety of AdePeCbl. (C) Residues interacting with the phosphodiester group in the nucleotide loop of cobalamin. Residues involved in coenzyme B 12 binding K i. Ogura et al. 6206 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS AdoCbl, and rapid inactivation during catalysis was not observed with these mutants (Fig. 3B). In contrast, k cat of the Kb135E mutant was only 2% that of the wild-type enzyme, and the k cat ⁄ k inact value indicated that this mutant underwent inactivation after 5.4 · 10 4 turnovers on average. These results indicate that a positive charge in the b135 residue is not absolutely required for catalytic activity, but the introduction of a negative charge there greatly lowers the activity. The former suggests that the salt bridge between the phos- phate group of the coenzyme nucleotide loop moiety and the side chain of Lysb135 is not essential for activ- ity. The latter would be probably due to the electro- static repulsion between the negatively charged phosphate group and –COO ) in the side chain of the b135 residue. The effects of Lysb135 mutations on K m values for AdoCbl and 1,2-propanediol as well as K i for CN-Cbl are summarized in Tables 1 and 2. For the Kb135R mutant, K m for AdoCbl and K i for CN-Cbl were rather smaller than those of the wild-type enzyme. This indicates that cobalamins are bound to this mutant more tightly than to the wild-type enzyme, probably because the salt bridge formation between the cobala- min phosphate group and the guanidinium group of Arg in the Kb135R mutant is appropriate. For the Kb135A and Kb135Q mutants, which have a neutral side chain at the b135 residue, K m values for AdoCbl and 1,2-propanediol and K i for CN-Cbl increased sig- nificantly. These results suggest that the affinities of the enzyme for cobalamins and substrate are lowered, probably due to the inability of these mutants to form a salt bridge with the phosphate group of cobalamins. In contrast, the Kb135E mutant, which has a negative charge at the b135 residue, showed a K m for AdoCbl and a K i for CN-Cbl that were larger than those of the wild-type enzyme by two orders of magnitude, although K m for 1,2-propanediol was comparable to the values of the Kb135A and Kb135Q mutants. It can therefore be concluded that the positive charge in the b135 residue is not essential for catalysis and is A B Fig. 2. PAGE analysis of the purified preparations of mutant diol de- hydratases. (A) SDS ⁄ PAGE. (B) Nondenaturing PAGE. Samples were electrophoresed on 11% (A) and 7% (B) polyacrylamide gels, and the resulting gels were subjected to protein staining with Coomassie Brilliant Blue R-250. Molecular mass markers, SDS-7 (Sigma-Aldrich, St Louis, MO, USA). BPB, bromophenol blue; wt, wild-type enzyme. The bands of the a-, b- and c-subunits are indicated on the right (A). The position of the (abc) 2 complexes is indicated on the right (B). Table 1. Kinetic parameters of mutant diol dehydratases, determined at 37 °C. The k cat values were determined by the alcohol dehydroge- nase–NADH coupled method using 1,2-propanediol as substrate. The k inact values were calculated from a change in the slope of a tangent to the time course curve of the reaction. The K m values were determined by the 3-methyl-2-benzothiazolinone hydrazone method. Aver- age ± standard deviation (n = 3). The AdoCbl concentrations used were 15 l M for the wild-type enzyme, Sa224A mutant and Kb135R mutant, 45 l M for the Sa224N mutant, 30 lM for the Kb135A mutant, 57 lM for the Kb135Q mutant, and 150 l M for the Kb135E mutant. Enzyme k cat ,s )1 (%) K m for 1,2-propanediol (m M) k cat ⁄ K m · 10 )6 (s )1 ÆM )1 ) k inact (min )1 ) k cat ⁄ k inact · 10 )4 Wild-type 336 (100) 0.15 ± 0.02 2.2 0.027 75 Sa224A 64 (19) 0.15 ± 0.01 0.43 0.46 0.8 Sa224N 17 (5) 1.90 ± 0.01 0.009 0.070 1.5 Kb135R 254 (76) 0.12 ± 0.01 2.1 0.027 56 Kb135A 196 (58) 0.39 ± 0.02 0.50 0.059 20 Kb135Q 211 (63) 0.39 ± 0.01 0.54 0.068 19 Kb135E 7.7 (2) 0.40 ± 0.06 0.019 0.0085 5.4 K i. Ogura et al. Residues involved in coenzyme B 12 binding FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6207 only moderately important for cobalamin binding, but the introduction of a negative charge largely prevents the enzyme–cobalamin interaction. Spectral changes of AdoCbl upon incubation with mutant enzymes in the presence of substrate Figure 4 shows the spectral changes of AdoCbl upon incubation with mutant apoenzymes in the presence of 1,2-propanediol. AdoCbl underwent a spectral change to cob(II)alamin (B 12r ) upon incubation with the wild- type or Sa224A apoenzyme for 5 min – that is, the absorbance at 525 nm decreased and a new peak at 478 nm appeared (Fig. 4A,B). These spectra reflect the steady-state concentrations of cob(II)alamin during catalysis. With the wild-type enzyme, it was not so much different from the spectrum obtained at 30 min of incubation. In the case of the Sa224A mutant, how- ever, the peak at 478 nm increased gradually upon prolonged incubation, and the spectrum obtained at 30 min of incubation resembled the typical spectrum of cob(II)alamin. As the Sa224A holoenzyme was completely inactivated by 10 min of incubation, this spectrum should be that of the completely inactivated holoenzyme of this mutant. The cob(II)alamin-like spe- cies in the inactivated Sa224A holoenzyme was stable even under aerobic conditions, but underwent oxida- tion to aquacobalamin (aqCbl) upon denaturation of the complex with guanidine-HCl under acidic condi- tions. The spectrum thus obtained no longer changed upon photoillumination, suggesting that the Co–C bond of the coenzyme had been completely and irre- versibly cleaved upon incubation with the Sa224A mutant for 30 min in the presence of substrate. In con- trast, the spectral change of AdoCbl upon incubation with the Sa224N apoenzyme was rather small even at 30 min of incubation (Fig. 4C). The spectrum observed after denaturation of this mutant holoenzyme was sim- ilar to that of free AdoCbl and changed to that of aqCbl upon photoillumination. Relatively low activity and a small k inact of the Sa224N mutant (Table 1) would account for the lower steady-state concentration of cob(II)alamin species and the slower rate of irre- versible cleavage of the Co–C bond with this mutant. When AdoCbl was incubated with the Kb135A mutant in the presence of substrate, a similar spectral change was observed within 5 min (Fig. 4D). However, the peak at 478 nm then decreased gradually, and the absorbance at 356 nm and  530 nm increased with time of incubation. These absorption peaks are characteristic of the diol dehydratase-bound Fig. 3. Time courses of 1,2-propanediol dehydration by mutant diol dehydratases. (A) Sera224 mutants. (B) Lysb135 mutants. The alco- hol dehydrogenase–NADH coupled method was used. The reaction mixture consisted of 60 ng of the wild-type or 600 ng of a mutant apoenzyme, 0.1 M 1,2-propanediol, 50 lg of yeast alcohol dehydro- genase, 0.2 m M NADH, 0.04 M potassium phosphate buffer (pH 8.0), and AdoCbl, in a total volume of 1.0 mL. The reaction was started by adding AdoCbl at a concentration given in the legend to Table 1. The absorbance changes (DA at 340 nm) per 60 ng of enzyme are shown here. Table 2. Binding affinities of mutant diol dehydratases for AdoCbl and CN-Cbl, determined at 37 °C. The k cat values were obtained as described in the legend to Table 1. Apparent K m and K i values were determined by the 3-methyl-2-benzothiazolinone hydrazone method, followed by Lineweaver–Burk plots. Average ± standard deviation (n = 3). Enzyme K m for AdoCbl (l M) k cat ⁄ K m (AdoCbl) · 10 )8 (s )1 ÆM )1 ) K i for CN-Cbl (l M) Wild-type 0.94 ± 0.11 3.6 1.5 ± 0.1 Sa224A 0.36 ± 0.05 1.8 0.63 ± 0.00 Sa224N 3.4 ± 0.2 0.05 2.3 ± 0.1 Kb135R 0.25 ± 0.04 10.2 0.51 ± 0.08 Kb135A 2.2 ± 0.4 0.89 1.4 ± 0.2 Kb135Q 5.4 ± 0.6 0.39 4.4 ± 0.5 Kb135E 127 ± 11 0.00061 463 ± 41 Residues involved in coenzyme B 12 binding K i. Ogura et al. 6208 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS hydroxocobalamin (OH-Cbl) [30], suggesting that the oxidation of cob(II)alamin accompanies the inactiva- tion of the Kb135A holoenzyme during catalysis. Upon denaturation of the complex after 30 min of incubation, the spectrum of enzyme-bound OH-Cbl was at least partly converted to a free aqCbl-like spec- trum. When the mixture was then photoilluminated, the spectrum underwent a further change to that of free aqCbl. It is therefore likely that AdoCbl was mostly converted to the enzyme-bound OH-Cbl by 30 min of incubation, but a fraction of the coenzyme still remained as AdoCbl at this time. EPR spectra obtained with the Sa224A mutant diol dehydratase When the wild-type holoenzyme was incubated with 1,2-propanediol at 4 °C for 1 min under anaerobic conditions, the typical EPR spectrum of reacting holo- enzyme was obtained (Fig. 5A). The characteristic high-field doublet signal with a splitting 14.3 mT was assigned to the 1,2-propanediol-1-yl radical (substrate-derived radical) [31], and the low-field broad signal to the low-spin Co(II) of cob(II)alamin. Such a spectrum arises from weak coupling in the Co(II)– organic radical pair [32–34]. The intensity of the dou- blet signal decreased within 3 min of incubation at 25 °C, and a new small peak with a g-value of  2.1 appeared upon further incubation for 30 min. The latter low-field signal might be due to the inactivated holoenzyme being formed. In contrast, only a trace of the typical doublet signal of reacting holoenzyme was observed with the Sa224A mutant upon incubation with substrate at 4 °C for 1 min (Fig. 5B). This indi- cates that the steady-state concentration of an organic radical intermediate is very low with the Sa224A mutant, which is consistent with the low catalytic activity of this mutant. Upon further incubation at 25 °C for 3 min, the trace of doublet signal disap- peared, and new signals with g-values of 2.08 and  2.2 appeared. Although the radical species giving the g = 2.08 signal has not yet been identified, the relative intensity of this signal increased with time of incubation, and the signal became predominant after Fig. 4. Spectral changes of AdoCbl upon aerobic incubation with mutant diol dehydratases in the presence of 1,2-propanediol. Apoenzyme (5 nmol) of the wild-type (A), Sa224A mutant (B), Sa224N mutant (C) or Kb135A mutant (D) was incubated at 30 °C with 4.5 nmol of AdoCbl in 0.01 M potassium phosphate buffer (pH 8.0) containing 1.3 M 1,2-propanediol and 1% Brij35, in a total volume of 1.0 mL. Spectra were taken at 5 min (thick solid lines) and 30 min (thin solid lines) after the addition of AdoCbl. Enzymes were then denatured by adding 6 M gua- nidine–HCl and 0.06 M citric acid. After incubation at 37 °C for 10 min, the mixture was neutralized to pH 8 by adding 200 lLof1M potas- sium phosphate buffer (pH 8.0) and 40 lLof5 M KOH. After the spectral measurement (broken lines), the mixture was photoilluminated in an ice-water bath for 10 min with a 300 W tungsten light bulb from a distance of 20 cm, and the spectra were taken again (dotted lines). Spectra are corrected for dilution. The spectra of apoenzymes were subtracted from the spectra obtained. K i. Ogura et al. Residues involved in coenzyme B 12 binding FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6209 33 min. It might be due to the same radical species as appeared with the wild-type enzyme upon prolonged incubation. The signal with a g-value of  2.2 might be assigned to Co(II) of cob(II)alamin. These results indicate that the Sa224A mutant undergoes rapid and irreversible inactivation during catalysis by the extinc- tion of an organic radical intermediate through unde- sirable side reaction(s). Fate of the adenosyl group of AdoCbl in inactivation of a mutant holoenzyme during catalysis To study the fate of the adenosyl group, the upper axial ligand of AdoCbl, in the inactivation of the Sa224A holoenzyme during catalysis, adenosyl group- derived product(s) from AdoCbl were identified. After 30 min of incubation of the Sa224A holoenzyme with 1,2-propanediol, the inactivated holoenzyme was dena- tured, and product(s) formed from the coenzyme were extracted and analyzed by HPLC on a reversed-phase column. The only nucleoside product derived from the adenosyl group was identified as 5¢-deoxyadenosine. The retention time of 5¢-deoxyadenosine was  8 min under the conditions employed. The formation of adenine, adenosine, 4¢,5¢-anhydroadenosine, 5¢,8-cyclic adenosine or adenosine 5¢-aldehyde was not observed at all. It is therefore evident that the inactivation of this mutant enzyme during catalysis is a mechanism- based one, because the hydrogen abstraction from sub- strate by the coenzyme adenosyl radical takes place as the initial event of catalysis. The amount of 5¢-deoxy- adenosine formed from 4.6 nmol of the Sa224A mutant and 15 nmol of AdoCbl was 4.7 nmol, which corresponds to approximately one mol per mol of enzyme. As diol dehydratase exists as a dimer of heterotrimers, this result suggests that only one of the two heterotrimeric units is involved in the formation of 5¢-deoxyadenosine. Recovery of active apoenzyme by resolution of an inactivated mutant enzyme A typical result of resolution experiments is shown in Table 3. The Sa224A mutant was completely inacti- vated by incubation with 1,2-propanediol for 30 min, followed by dialysis. After resolution by acid ammo- nium sulfate treatment, 56% of the original specific activity of the mutant enzyme was recovered. The reso- lution of cobalamin by this procedure was not com- plete, and the resolved enzyme still contained OH-Cbl. The cobalamin recovered in the supernatant was aqCbl, and the extent of cobalamin resolution was Fig. 5. EPR spectra observed upon incubation of the wild-type (A) and Sa224A mutant (B) holodiol dehydratases with 1,2-propanediol. The arrows correspond to g = 2.0. Holoenzymes were formed under an argon atmosphere by incubating 1.9 mg (9.2 nmol) of sub- strate-free wild-type and Sa224A apoenzymes at 25 °C for 3 min with 50 nmol of AdoCbl in 0.65 mL of 0.05 M potassium phosphate buffer (pH 8.0) containing 18 m M sucrose monocaprate. The enzyme reaction was started by adding 50 lmol of 1,2-propanediol in 0.05 mL. After 1 min at 4 °C, the reaction mixture was rapidly frozen in an isopentane bath that had been previously cooled to approximately )160 °C, and then in a liquid nitrogen bath. EPR spectra were taken at )130 °C. After the first measurement, the mixture was incubated at 25 °C for 3 min and frozen again, as described above, for the second measurement. The mixture was then incubated at 25 °C for an additional 5 min and 25 min for the third and fourth measurements. Residues involved in coenzyme B 12 binding K i. Ogura et al. 6210 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS estimated to be 61% from the cobalamin contents of the 1,2-propanediol-inactivated Sa224A holoenzyme and of the resolved enzyme. This value of cobalamin resolution is in good agreement with the recovery of enzyme activity. It was therefore concluded that resolved apoenzyme recovered from the inactivated mutant holoenzyme could be reconstitutable to fully active holoenzyme – that is, the Sa224A apoenzyme itself did not undergo damage in the mechanism-based inactivation by the substrate 1,2-propanediol. Inactivation of mutant holoenzymes by O 2 in the absence of substrate The holoenzyme of diol dehydratase undergoes irre- versible inactivation by O 2 in the absence of substrate [35]. This inactivation is accompanied by the irrevers- ible Co–C bond cleavage of the enzyme-bound coen- zyme, forming OH-Cbl. It is thus believed that the inactivation is caused by the reaction of the activated Co–C bond with O 2 , and thus reflects the extent of Co–C bond activation upon the coenzyme binding to apoenzyme in the absence of substrate. As shown in Table 4, the inactivation followed pseudo-first-order reaction kinetics, with a rate constant (k inact,O2 )of 0.20 min )1 for the wild-type holoenzyme. To examine the contributions of enzyme–coenzyme interactions at the Sera224 and Lysb135 residues to Co–C bond acti- vation, rates of O 2 inactivation of mutant holoenzymes in the absence of substrate were determined. The rate constants of O 2 inactivation for the Lysb135 mutants were in the range 0.13–0.17 min )1 , which is slightly slower but almost comparable to that of the wild-type holoenzyme. In contrast, the rate of O 2 inactivation of mutant holoenzymes was very slow for the Sa224A mutant, and inactivation was not observed with the Sa224N mutant. These results suggest that the appro- priate hydrogen bonding between the side chain OH group of Sera224 and N3 of the adenine ring of the coenzyme adenosyl group is important for activation of the Co–C bond in the absence of substrate as well. On the other hand, ion pairing between the e-NH 2 group of Lysb135 and the phosphate group of the coenzyme nucleotide loop seems not to be essential for Co–C bond activation. This would be reasonable, because the cobalamin moiety of AdoCbl is accommo- dated to the cobalamin-binding site of the enzyme through multiple interactions with many amino acids. Discussion The homolytic fission of the Co–C bond of enzyme- bound AdoCbl leads to the introduction of an adenosyl radical, a catalytic radical, into the active sites. This is an essential early event in all of the AdoCbl-dependent enzymatic reactions [1–3,7,8]. We synthesized various coenzyme analogs in which one of the structural compo- nents is substituted by a closely related group, and used them as probes to investigate the mechanism of enzy- matic activation (labilization) of the coenzyme Co–C bond as well as the role of each structural component of the coenzyme in the interaction with diol dehydratase [1,11–17]. It was demonstrated that the cobalamin moi- ety [14–17,29] and the adenosyl group [11–13] are required for its tight binding to the apoenzyme and for activation of the Co–C bond, respectively, and that the ‘adenine-attracting effect’ of the apoenzyme is a major element that weakens the Co–C bond [36,37]. Later, the X-ray structures of the diol dehydratase–AdePeCbl complex revealed that the enzyme has a cobalamin-bind- ing site [18] and an adenine-binding pocket [21] for Ado- Cbl. A modeling study using X-ray structures suggested that the tight binding of AdoCbl to both of these sites induces marked distortions, including both angular strains and tensile force, that inevitably lead to Co–C bond cleavage [21,38]. We proposed this ‘steric strain model’ as the molecular mechanism for the enzymatic activation of the coenzyme’s Co–C bond. The X-ray structures of diol dehydratase show that the coenzyme adenine moiety is anchored in the pro- tein by hydrogen bonding of N3 with the side chain OH group of Sera224, of 6-NH 2 and N7 with main chain amide groups of other residues, and of N1 with a water molecule [21]. Cobalamin is accommodated to a space that is mainly surrounded by hydrophilic groups [18]. Five amide groups out of six peripheral Table 3. Resolution of 1,2-propanediol-inactivated Sa2424A mutant holoenzyme by acid ammonium sulfate treatment. Specific activity, UnitsÆmg )1 (%) B 12 bound, l M(%) Apoenzyme used 2.5 (100) 0.0 (0) Inactivated holoenzyme 0.0 (0) 3.1 (100) Resolved enzyme 1.4 (56) 1.2 (39) Table 4. O 2 inactivation of mutant holo-diol dehydratases in the absence of substrate, determined at 37 °C. Enzyme k inact,O2 (min )1 ) Wild-type 0.20 Sa224A 0.03 Sa224N Not inactivated a Kb135R 0.17 Kb135A 0.16 Kb135Q 0.13 a No inactivation was observed for at least 10 min. K i. Ogura et al. Residues involved in coenzyme B 12 binding FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6211 side chains of the corrin ring form hydrogen bonds with five amino acids in the a-subunit and three in the b-subunit. The phosphate group of cobalamin forms a salt bridge between the e-amino group of Lysb135, in addition to hydrogen bonds with the two residues in the b-subunit. In this study, the impacts of adenine anchoring and ion pairing on catalysis were evaluated by site-directed mutagenesis at Sera224 and Lysb135. The Sa224A mutant, which cannot form a hydrogen bond with N3 of the adenine moiety, showed a relative activity (k cat ) of 19% and decreased sensitivity of the holoenzyme to O 2 in the absence of substrate, suggest- ing the importance of hydrogen bonding between N3 of the adenine moiety and the side chain OH group of Sera224 for activation of the coenzyme Co–C bond. On the other hand, the Sa224N mutant had only 5% relative activity, which was much lower than that of the Sa224A mutant. Its complex with AdoCbl did not show the sensitivity to O 2 in the absence of substrate. One possibility is that the hydrogen bond might not be formed or be formed at an improper position with the Sa224N mutant. The other possibility is that it might be due to the coenzyme-binding problems caused by the Sa224N mutation, regardless of the hydrogen bonding interaction with N3. The O 2 inactivation of the holoenzyme occurs only in the absence of substrate. The inactivation mechanism remains unclear at present, and the identification of the inactivation products would provide important clues to solve this problem. It was well established from the spec- tral changes that OH-Cbl is formed upon the inactiva- tion [35]. However, products from the adenosyl group of AdoCbl have not yet been definitely identified [35] (M. Yamanishi, S. Yamanaka & T. Toraya, unpub- lished results). Product(s) from O 2 also remain to be identified. O 2 inactivation can be considered to be clo- sely related to catalysis, because only the complexes with active coenzyme analogs undergo this inactivation, except for the complexes with 3-deazaAdoCbl [12], neb- ularylcobalamin (deamino analog of AdoCbl) [11], and aristeromycylcobalamin (carbocyclic analog of AdoCbl) [11,39]. The substrate facilitates Co–C bond cleavage by inducing conformational changes that increase the steric strain of the Co–C bond of enzyme-bound coenzyme [38]. Therefore, these results suggest that the Co–C bond activation in the absence of substrate is not sufficient with coenzyme analogs that lack hydrogen bonding interactions with the enzyme. The k cat ⁄ k inact value is a good measure of the resis- tance of holoenzymes to mechanism-based inactivation. These ratios for the Sa224A and Sa224N mutants indi- cate that these enzymes underwent inactivation after only 8000 and 15 000 turnovers, respectively, on aver- age. We have previously reported that the wild-type enzyme shows a small k cat ⁄ k inact value when 3-dea- zaAdoCbl, a coenzyme analog that cannot form a hydrogen bond with Sera224, is used as coenzyme [12]. It can thus be concluded that the proper hydrogen bonding between N3 of the adenine moiety and Sera224 plays an essential role in protecting highly reactive radi- cal intermediate(s) from undesired side reactions, proba- bly through stable anchoring of the adenine moiety to the adenine-binding pocket. Upon the inactivation of Sa224A, a cob(II)alamin-like spectrum was observed. The EPR spectrum suggested that cob(II)alamin and an unidentified radical species were formed from AdoCbl and accumulated upon prolonged incubation with the Sa224A mutant. A stoichiometric amount of 5¢-deoxy- adenosine was formed upon inactivation from the enzyme-bound coenzyme, but the apoenzyme itself was not damaged. Thus, the inactivation of the Sa224A mutant during catalysis was concluded to be a mecha- nism-based one, as shown below: AdoH AdoH E Ado Cbl III SH E Ado Cbl II SH • • E Cbl II •S • side reaction E Cbl II • where E is enzyme, SH is substrate, Cbl is cobalamin, and Ado is 5¢-deoxy-5¢-adenosyl. Mutant enzymes in which Lysb135 was substituted with Arg, Ala or Gln showed relatively high activity and relatively large k cat ⁄ k inact values, whereas the Kb135E mutant possessed a trace of activity and a small k cat ⁄ k inact value. Binding affinities for AdoCbl and CN-Cbl were strengthened when Lysb135 was substituted with Arg, being almost the same as the those of the wild-type enzyme upon the Ala substitu- tion, and slightly lowered upon the Gln substitution at the b135 residue. This might be due to hydrogen bond- ing and interactions other than the ion pairing being strong enough to maintain the tight binding of cobala- min. In contrast, the Kb135E mutant showed more than 100-fold lower affinity for both cobalamins. These results indicate that a salt bridge between the phosphate group and the side chain of the b135 resi- due is not essential for either catalysis or cobalamin binding, but the introduction of a negative charge in the b135 residue destroyed the affinity for cobalamins and lowered the enzyme activity to 2% even when AdoCbl was used at a concentration higher than its K m . It was also evident from the decreased k cat ⁄ k inact value with the Kb135E mutant that the electrostatic repulsion between the cobalamin phosphate group and the side chain –COO ) of Glub135 also results in the destabilization of the reactive radical intermediate(s) during catalysis. Residues involved in coenzyme B 12 binding K i. Ogura et al. 6212 FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS Experimental procedures Materials Crystalline AdoCbl was a gift from Eisai Co. Ltd (Tokyo, Japan). Crystalline CN-Cbl was obtained from Glaxo Research Ltd (Greenford, UK). Other chemicals were analyti- cal grade reagents and were used without further purification. Construction of expression plasmids for mutant diol dehydratases The mutations described in this article were introduced into the diol dehydratase genes (pddABC)ofKlebsiella oxytoca (formerly Aerobacter aerogenes) ATCC8724, using a Quik- Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). pUSI2E(DD) [22], an expression plasmid for the wild-type enzyme, was used as a template. The mutagenic sense primers used were 5¢-ctacgccgaaaccatc gccgtctacggcac-3¢ for Sa224A, 5¢-ctacgccgaaaccatc aacgtctacggcac-3¢ for Sa224N, 5¢-ggcatccagtcg agaggcaccacggtgatc-3¢ for Kb135R, 5¢-ggcatccagtcg gcaggcaccacggtgatc-3¢ for Kb135A, 5¢-ggcatc cagtcg caaggcaccacggtgatc-3¢ for Kb135Q, and 5¢-gcatc cagtcg gaaggcaccacggtgatc-3¢ for Kb135E, in which underlin- ing indicates amino acid substitutions. The oligonucleotides having the complementary sequences in the opposite direc- tion were used as the respective antisense primers. It was con- firmed by sequencing of the DNA region encompassing the entire diol dehydratase genes and tac promoter that no unin- tended mutations had been incorporated during mutagenesis. Expression and purification of mutant diol dehydratases E. coli XL1-Blue cells were transformed with the above-men- tioned expression plasmids. Recombinant E. coli cells were grown aerobically in LB medium containing 0.1% 1,2-pro- panediol and ampicillin (50 lgÆmL )1 ), and induced by 1 mm isopropyl b-d-1-thiogalactopyranoside, as described previ- ously [22]. Mutant apoenzymes were purified from over- expressing E. coli cells, essentially as described previously for the recombinant wild-type enzyme [28]. The DEAE–cellulose chromatography step was omitted, as the enzymes extracted from crude membrane fractions were found to be almost homogeneous. However, the DEAE–cellulose-purified apoenzymes were used in the EPR experiments. Substrate-free apoenzymes Substrate-free apoenzymes for the measurements of K m values for 1,2-propanediol and rates of inactivation of holoenzymes in the absence of substrate were obtained by dialysis at 4 °C for 36 h against 100 volumes of 50 mm potassium phosphate buffer (pH 8.0) containing 0.1% Brij35 with two buffer changes. Enzyme and protein assays Diol dehydratase activity was routinely measured by the 3-methyl-2-benzothiazolinone hydrazone method, using 1,2-propanediol as substrate [11]. One unit is defined as the amount of enzyme activity that catalyzes the formation of 1 lmol of propionaldehyde ⁄ min at 37 °C under the stan- dard assay conditions. Time courses of the diol dehydratase reaction were measured by the alcohol dehydrogenase– NADH coupled method [29]. The protein concentration of purified enzyme was deter- mined by measuring the absorbance at 280 nm. The molar absorption coefficient at 280 nm, calculated by the method of Gill & von Hippel [40], for diol dehydratase is 120 500 m )1 Æcm )1 [41]. PAGE PAGE analyses of purified mutant enzymes were performed under nondenaturing conditions as described by Davis [42], in the presence of 0.1 m 1,2-propanediol [22], and under denaturing conditions as described by Laemmli [43]. Pro- teins were stained with Coomassie Brilliant Blue R-250. EPR measurements The wild-type and the Sa224A mutant apoenzymes purified as described previously [28] were used. Substrate-free apoen- zyme solution [1.9 mg of protein in 0.6 mL of 50 mm potas- sium phosphate buffer (pH 8.0) containing 20 mm sucrose monocaprate] was mixed at 0 °C with AdoCbl solution (50 nmol in 0.05 mL) in a quartz EPR tube (outside diameter 5 mm) stoppered with a rubber septum. After replacement of the air in the tube with argon by repeated evacuation and flushing with argon three times, holoenzymes were formed, reacted with 1,2-propanediol, and rapidly frozen as described in the legend to Fig. 5. The frozen sample was transferred to the EPR cavity and cooled with a cold nitrogen gas flow con- trolled by a Eurotherm B-VT 2000 temperature controller. EPR spectra were taken as described previously [44,45] at )130 °C on a Bruker ESP-380E spectrometer modified with a Gunn diode X-band microwave unit. EPR microwave frequency was 9.484–9.488 GHz, modulation amplitude was 1 mT, modulation frequency was 100 kHz, and microwave power was 10 mW. Fate of the adenosyl group of AdoCbl in inactivation of mutant holoenzymes during catalysis The adenosyl group-derived product(s) formed from AdoCbl in the inactivation of a mutant holoenzyme during catalysis was identified as described previously [46]. Substrate-free apoenzyme (1.0 mg, 4.6 nmol) was incubated at 37 °C for 30 min in the dark with 15 lm AdoCbl in the presence of 0.1 m 1,2-propanediol. The enzyme protein was then K i. Ogura et al. Residues involved in coenzyme B 12 binding FEBS Journal 275 (2008) 6204–6216 ª 2008 The Authors Journal compilation ª 2008 FEBS 6213 [...]... sulfate fractionation and was no longer effective for solubilizing the enzyme Instead, trypsin-solubilized Sa224A apoenzyme was prepared as described previously [46] and successfully used in the resolution experiments The Sa224A holoenzyme inactivated during catalysis was obtained by the incubation of trypsinsolubilized apoenzyme (1.5 mg) at 37 °C for 30 min with AdoCbl with the substrate 1,2-propanediol,... determined spectrophotometrically O2 inactivation of holoenzymes in the absence of substrate Appropriate amounts of substrate-free wild-type and mutant apoenzymes were incubated aerobically with 72 lm AdoCbl at 37 °C for various time periods in 35 mm potassium phosphate buffer (pH 8.0) containing 50 mm KCl Substrate (0.1 m 1,2-propanediol) was then added to stop the O2 inactivation, and the remaining... (1986) The synthesis of adenine- modified analogs of adenosylcobalamin and their coenzymic function in the reaction catalyzed by diol dehydrase J Biol Chem 261, 9289–9293 13 Ushio K, Fukui S & Toraya T (1984) Coenzymic function of 1- or N6-substituted analogs of adenosylcobalamin in the diol dehydratase reaction Biochem Biophys Acta 788, 318–326 14 Toraya T & Ishida A (1991) Roles of the d-ribose and 5,6-dimethylbenzimidazole...Residues involved in coenzyme B12 binding K.-i Ogura et al denatured by adding ethanol to a final concentration of 80% The mixture was heated at 90 °C for 10 min, and then centrifuged at 8060 g for 10 min The supernatant was evaporated to a small volume and taken up into 0.5 mL 15% methanol containing 1% acetic acid The nucleoside product from the adenosyl group was analyzed by HPLC using a Cosmosil... followed by dialysis, and the inactivated holoenzyme obtained was then resolved by acid ammonium sulfate treatment, as described previously [47] The activities of the apoenzyme used, the inactivated holoenzyme and the resolved enzyme were measured by the alcohol dehydrogenase–NADH coupled method in the presence of added AdoCbl The amounts of cobalamin bound to the inactivated holoenzyme and the resolved enzyme... Role of peripheral side chains of vitamin B12 coenzymes in the reaction catalyzed by dioldehydrase Biochemistry 18, 417–426 30 Toraya T, Watanabe N, Ushio K, Matsumoto T & Fukui S (1983) Ligand exchange reactions of diol dehydrase-bound cobalamins and the effect of the nucleoside binding J Biol Chem 258, 9296–9301 31 Yamanishi M, Ide H, Murakami Y & Toraya T (2005) Identification of the 1,2-propanediol-1-yl... coordination of 5,6-dimethylbenzimidazole to the cobalt atom of adenosylcobalamin bound to diol dehydratase Biochemistry 37, 4799–4803 Abend A, Nitsche R, Bandarian V, Stupperich E & ´ Retey J (1998) Dioldehydratase binds coenzyme B12 in the ‘base-on’ mode: ESR investigations on cob(II)alamin Angew Chem Int Ed 37, 625–627 Masuda J, Shibata N, Morimoto Y, Toraya T & Yasuoka N (2000) How a protein generates... measured by incubating the mixtures at 37 °C for an additional 10 min Acknowledgements This work was supported in part by Grants -in- Aid for Scientific Research [(B) 13480195 and 17370038 and Priority Areas 753 to T Toraya, and (C) 14580627 to T Tobimatsu] from the Japan Society for Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Grant 6214 of Natural... 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 Residues involved in coenzyme B12 binding 26 Seyfried M, Daniel R & Gottschalk G (1996) Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of. .. an intermediate in adenosylcobalamin-dependent diol dehydratase reaction Biochemistry 44, 2113–2118 32 Schepler KL, Dunham WR, Sands RH, Fee JA & Abeles RH (1975) A physical explanation of the EPR spectrum observed during catalysis by enzymes utilizing coenzyme B12 Biochim Biophys Acta 397, 510–518 33 Buettner GR & Coffman RE (1977) EPR determination of the Co(II)-free radical magnetic geometry of the . Roles of adenine anchoring and ion pairing at the coenzyme B 12 -binding site in diol dehydratase catalysis Ken-ichi Ogura, Shin-ichi Kunita,. [18]. In this article, we report the roles of adenine anchor- ing and ion pairing at the AdoCbl-binding site in diol dehydratase catalysis. To study the