Interactions between coenzyme B 12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum Hao-Ping Chen 1 , Huei-Ju Hsu 1 , Fang-Ciao Hsu 1 , Chien-Chen Lai 2 and Chung-Hua Hsu 3 1 Institute of Biotechnology, National Taipei University of Technology, Taiwan 2 Institute of Molecular Biology, National Chung-Hsing University, Taichung, Taiwan 3 Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan Glutamate mutase from Clostridium tetanomorphum is one of a group of adenosylcobalamin (AdoCbl)-depen- dent mutases that catalyzes the inter-conversion of l-glutamate and threo-b-methyl-l-aspartate. It com- prises two weakly-associating subunits, MutS and MutE, which combine with AdoCbl to form the active holo-enzyme [1]. The coenzyme is known to be bound by glutamate mutase in ‘base-off ⁄ His-on’ mode [2]. As shown in Fig. 1A, the lower axial ligand of the cobalt atom, 5,6-dimethylbenzimidazole, is replaced by a his- tidine residue within a conserved B 12 -binding motif, DXHXXG(14–19). Model studies have shown that the cobalt–carbon bond dissociation energy of the cofactor is sensitive to changes in the pK a of the lower axial base [3]. This has led to speculation that proteins might modulate the pK a of the histidine via the hydrogen bond between the His–Asp pair and so ‘fine tune’ the reactivity of AdoCbl. Mutations of either residue result in significant impairment of the protein’s coenzyme-binding ability, as well as its catalytic ability [4]. The biosynthesis of AdoCbl is a very complicated process. 5¢-deoxyadenosyl- cobinamide (AdoCbi) and AdoCbi-GDP are intermediates during the biosynthesis of AdoCbl (Fig. 2A). Previous studies have shown that AdoCbl-dependent methylmalonyl CoA mutase binds both coenzyme analogs in ‘base-off’ mode, which indi- cates that the histidine residue located on the conserved cobalamin-binding motif is unable to coordinate to the cobalt atom [5,6]. However, the AdoCbi-GDP-reconsti- Keywords adenosylcobalamin; adenosylcobinamide; AdoCbi-GDP; B 12 ; glutamate mutase Correspondence H P. Chen, Institute of Biotechnology, National Taipei University of Technology 1, Sec 3, Chung-Hsiao East Road, Taipei 106, Taiwan Fax: +886 2 27317117 Tel: +886 2 27712171 ext. 2528 E-mail: hpchen@ntut.edu.tw (Received 14 August 2008, revised 30 September 2008, accepted 2 October 2008) doi:10.1111/j.1742-4658.2008.06724.x Adenosylcobalamin (AdoCbl)-dependent glutamate mutase from Clostrid- ium tetanomorphum comprises two weakly-associating subunits, MutS and MutE, which combine with AdoCbl to form the active holo-enzyme. Three coenzyme analogs, methylcobinamide (MeCbi), adenosylcobinamide (Ado- Cbi) and adeosylcobinamide-GDP (AdoCbi-GDP), were synthesized at milligram scale. Equilibrium dialysis was used to measure the binding of coenzyme B 12 analogs to glutamate mutase. Our results show that, unlike AdoCbl-dependent methylmalonyl CoA mutase, the ratio k cat ⁄ K m decreased approximately 10 4 -fold in both cases when AdoCbi or AdoCbi- GDP was used as the cofactor. The coenzyme analog-binding studies show that, in the absence of the ribonucleotide tail of AdoCbl, the enzyme’s active site cannot correctly accommodate the coenzyme analog AdoCbi. The results presented here shed some light on the cobalt–carbon cleavage mechanism of B 12 . Abbreviations AdoCbi, adenosylcobinamide; AdoCbl, adenosylcobalamin; Ado-PCC, (Cob-5¢-Deoxyadenosin-5¢-yl)-(p-cresyl)cobamide; (Bza)AdoCba, (benzimidazolribofuranosyl)-adenosylcobinamide; CobU, adenosyl-cobinamide kinase ⁄ adenosyl-cobinamide-phosphate guanylyltransferase; MeCbi, methylcobinamide. 5960 FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS tuted enzyme is catalytically active. More importantly, the k cat ⁄ K m of methylmalonyl CoA mutase is only four-fold lower when AdoCbi-GDP is used as cofactor [5,6]. This unexpected result suggests that coordination by the lower axial ligand is not essential in the case of methylmalonyl CoA mutase. To study the reactivity of glutamate mutase toward these coenzyme analogs, a chemo-enzymatic method was developed to synthesize AdoCbi-GDP at the milligram scale. Our results show that, in contrast to methylmalonyl CoA mutase, neither AdoCbi nor AdoCbi-GDP can efficiently act as cofac- tor for glutamate mutase [5]. The binding of AdoCbl and three coenzyme analogs, methylcobinamide (MeC- bi), AdoCbi and AdoCbi-GDP, to glutamate mutase was measured by equilibrium dialysis. Kinetic proper- ties towards AdoCbi and AdoCbi-GDP were also investigated. Here, we report the results of coenzyme- binding and kinetic studies of AdoCbl analogs with glutamate mutase. Results Synthesis of MeCbi, AdoCbi and AdoCbi-GDP MeCbi and AdoCbi were successfully separated from unreacted MeCbl and AdoCbl and the dealkylated side products using an SP–Sepharose ion-exchange column. The relative molecular masses of MeCbi and AdoCbi determined by ESI-MS were 1004.5 and 1240, which compare favorably with calculated relative molecular masses for MeCbi and AdoCbi of 1004.1 and 1239.6, respectively. The bifunctional enzyme CobU (adenosyl- cobinamide kinase ⁄ adenosyl-cobinamide-phosphate guanylyltransferase) is involved in biosynthesis and assembly of the nucleotide loop of cobalamin [7,8] (Fig. 2A,B) Using chemically synthesized AdoCbi as the CobU substrate, AdoCbi-GDP was enzymatically pre- pared in large quantities. The yield of AdoCbi-GDP could be significantly enhanced by using phenol ⁄ dichlo- romethane extraction to remove the salt component of the AdoCbi solution. The recovery of AdoCbi-GDP by reverse-phase HPLC was very reproducible (Fig. 3). The relative molecular mass of AdoCbi-GDP determined by ESI-MS was 1664.4, and the calculated relative molecu- lar mass of AdoCbi-GDP is 1664.6. The HPLC method that we developed in this study is quite straightforward, separating AdoCbi and AdoCbi-GDP directly without further modification. In contrast, the reactant and prod- uct, AdoCbi and AdoCbi-GDP, were analyzed in the form of (CN) 2 Cbi and (CN) 2 Cbi-GDP, respectively, in previous reports [7,8]. 1 H-NMR spectra for MeCbi and AdoCbi have been published previously [9,10]. The 600 MHz NMR spectrum of AdoCbi-GDP in D 2 O ⁄ H 2 O was analyzed using two-dimensional COSY and NOESY experiments. The results are summarized in Table 1 and Fig. 2B. D14 C15 H16 G120 T121 S61 V60 L59 G92 G91 Y117 I22 L23 A118 I334 E330 T94 R66 A67 G68 E subunit (53.7 kDa) S subuniT (14 kDa) H610 D608 G609 G686 G685 G613 G653 V654 S655 Y705 T709 T706 I617 E370 E247 Y243 Y89 Q330 L374 AB Fig. 1. (A) Model of glutamate mutase showing AdoCbl bound between the MutS and MutE subunits. The coenzyme-binding domain is on the MutS subunit. (B) Model of methylmalonyl CoA mutase. The AdoCbl molecule is shown in grey and protein residues are shown in black. H P. Chen et al. Adenosylcobalamin-dependent glutamate mutase FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS 5961 O NH 2 CONH 2 CONH 2 H 2 NOC H 2 NOC CONH 2 O OH NH H N N N N NH 2 O OH HO HH Co N N N N AdoCbi O NH 2 CONH 2 CONH 2 H 2 NOC H 2 NOC CONH 2 O O - P O HN H O O N N N N NH 2 O OH HO HH Co N N N N N N N N NH 2 O OH O O - P O O AdoCbi-GDP O NH 2 CONH 2 CONH 2 H 2 NOC H 2 NOC CONH 2 O NH H 3 C H N N N N NH 2 O OH HO HH Co N N N N AdoCbi-P P O O O - O ATP ADP GTP PP i Cobinamide kinase Cobinamide kinase O NH 2 CONH 2 CONH 2 H 2 NOC H 2 NOC CONH 2 O N N O OH HO O P O NH H O O - N N N N NH 2 O OH HO HH Co N N N N AdoCbl α-ribazole GMP Cobalamin synthase A Pr AdoCbi-GDP N N NN Co NH 2 O H 2 N H 2 N O NH 2 O O NH 2 O NH 2 O NH O P O P O O O N OH N N NH O NH 2 R 5 4 6 3 2 1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 25 26 27 30 31 32 35 37 36 38 41 42 46 47 48 49 50 53 54 55 56 57 60 61 Pr1 Pr2 3 R2 R3 R4 R5 R= N N N N O OH OH H 2 C NH 2 A15 A14 A13 A12 A11 O - O - O O R1 A2 A8 a b c d 43 e f g B2 A4 A5 B Fig. 2. (A) Schematic representation of the final steps of the de novo AdoCbl biosyn- thetic pathway. (B) The chemical structure of AdoCbi-GDP. Adenosylcobalamin-dependent glutamate mutase H P. Chen et al. 5962 FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS Determination of dissociation constants for cofactors by equilibrium dialysis The binding of AdoCbl, MeCbi, AdoCbi and AdoCbi- GDP to glutamate mutase was investigated by equilib- rium dialysis. Figure 4 shows the analog binding curves with a fixed concentration of glutamate mutase. AdoCbl, MeCbi, AdoCbi and AdoCbi-GDP were bound with apparent K d values of 3.7 ± 0.5, 6.0 ± 0.9, 18 ± 3 and 14 ± 3 lm, respectively (Fig. 4A–D). UV–visible spectra of protein-bound MeCbi, AdoCbi and AdoCbi-GDP complexes The UV–visible spectra of cobalamins provide a useful tool to examine the coordination state of cobalt. The UV–visible absorption spectra of the MeCbi-glutamate mutase, AdoCbi-glutamate mutase and AdoCbi-GDP-glutamate mutase complexes were measured. A red shift was observed in the spectra of protein-bound MeCbi, AdoCbi and AdoCbi-GDP. The 522 nm absorption maximum suggests that the histidine residue occupies the lower axial ligand posi- tion of the cobalt atom. However, we estimate that approximately 55–60% of the AdoCbi–glutamate mutase complex binds the cofactor in the ‘His-on’ form (Fig. 5). 150 A B AdoCbi 100 50 0 150 100 50 0 0 5 10 15 20 Time 25 30 35 40 45 AdoCbi-GDP 300 200 100 0 300 200 100 0 0 5 10 15 20 Tim e 25 30 35 40 45 Fig. 3. Purification of AdoCbi-GDP from the reaction mixture by reverse-phase HPLC. (A) Before the CobU enzymatic reaction. (B) After the CobU enzymatic reaction. Table 1. 600 MHz 1 H-NMR data for AdoCbi-GDP. d, doublet; q, quadruplet; s, singlet; t, triplet; td, triplet of doublets; dd, doublet of doublets. Assignment Signal type Chemical shifts AdoCbi-GDP (pH 7.0, 25 °C) (p.p.m.) J couplings (AdoCbi-GDP) (Hz) Corrin methyl C20 s 0.77 C25 s 1.38 C35 s 2.38 C36 s 1.79 C46 s 0.83 C47 s 1.57 C53 s 2.36 C54 s 1.12 Corrin CH C3 m 4.19 C8 m 3.76 C10 s 6.92 C13 dd 3.35 5.77, 3.23 C18 td 2.8 10.4, 3.58 C19 d 4.62 3.58 Corrin CH 2 side chain C26 d 2.62, 2.3 14.6 C30 m 1.96, 1.85 C31 m 2.44 C37 d 2.22, 1.74 14.8 C41 m 1.93, 1.81 C42 m 2.32, 2.24 C48 m 1.92, 1.77 C49 m 2.16 C55 m 1.75 C56 m 2.27 C60 d 2.63, 2.40 10.4 Aminopropan-2- ol side chain Pr1(CH 2 ) t 3.30, 3.185 5.5 Pr2(CH) m 4.38 Pr3(CH 3 ) d 1.21 6.5 Loop ribose R1 d 5.85 6.81 R2 m 4.68 R3 dd 4.46 4.87, 3.47 R4 m 4.27 R5 m 4.15 Adenosyl A2 s 8.20 A8 s 8.02 A11 d 5.60 3.54 A12 dd 4.40 5.54, 5.75 A13 dd 3.75 6.54, 5.75 A14 dd 1.91 9.3, 6.54 A15 d, dd 0.50, 0.32 8.6;8.6, 9.3 Base B2 s 8.01 NH s 8.20 s 7.94 s 7.84 s 7.6 s 7.57 s 7.3 s 7.27 s 7.09 s 7.02 s 6.88 s 6.86 s 6.60 s 6.57 s 6.36 H P. Chen et al. Adenosylcobalamin-dependent glutamate mutase FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS 5963 Enzyme assay In order to investigate the role of the ribonucleotide tail of AdoCbl in catalysis, the coenzyme analogs were used to examine the enzymatic activity. Our results indicate that, perhaps not surprisingly, MeCbi is a totally inac- tive coenzyme. The K m values for AdoCbi and Ado- Cbi-GDP were 26 ± 8 and 75 ± 28 lm, respectively, and the k cat values were (9.8 ± 1.0) · 10 )3 Æs )1 and (4.5 ± 0.8) · 10 )3 Æs )1 , respectively. In both cases, the k cat ⁄ K m was decreased by approximately 10 4 -fold compared with that of AdoCbl. Discussion Both methylmalonyl CoA mutase and glutamate mutase belong to the subfamily of B 12 -dependent car- bon-skeleton mutases, but their 1,2-rearrangement mechanisms are obviously different [11]. Previous studies have shown that (a) AdoCbi does not support the turnover of methylmalonyl CoA mutase, but Ado- Cbi-GDP does, and (b) the enzyme binds both AdoCbi and AdoCbi-GDP in ‘base-off ⁄ His-off’ mode. The results presented here indicate that, in contrast to methylmalonyl CoA mutase, the k cat ⁄ K m of glutamate mutase for both analogs decreased by approximately 10 4 -fold. These results suggest that the ribonucleotide tail of AdoCbl plays an important role in catalysis in the case of glutamate mutase. In addition, both cofac- tor analogs tested are bound by glutamate mutase in ‘base-off ⁄ His-on’ mode. Histidine–cobalt ligation therefore cannot efficiently facilitate turnover of the enzyme in the absence of the ribonucleotide tail of AdoCbl. It is apparent that glutamate mutase is mech- anistically different from methylmalonyl CoA mutase. Significant differences in the affinity for AdoCbl between these two enzymes appear to exit. Methylmal- onyl CoA mutase binds AdoCbl very tightly with a K d of 0.17 lm, while glutamate mutase binds AdoCbl relatively weakly with a K d between 1.8 and 6.8 lm [1]. Moreover, glutamate mutase is very sensitive to perturbation of the cofactor’s nucleotide tail, while methylmalonyl CoA mutase is not. (Benzimidazolribo- furanosyl)-adenosylcobinamide [(Bza)AdoCba] is a coenzyme B 12 analog in which the dimethylbenzimi- dazole moiety of AdoCbl is replaced by benzimidazole. Previous studies have shown that the apparent K m of glutamate mutase for (Bza)AdoCba is 0.5 lm, while that for AdoCbl is 18 lm under similar conditions [12]. However, the only difference between AdoCbl and (Bza)AdoCba is two methyl groups. In contrast, (Co-b-5¢-Deoxyadenosin-5¢-yl)-(p-cresyl)cobamide (Ado- PCC) is another ‘base-off’ coenzyme B 12 analog in which the dimethylbenzimidazole moiety of AdoCbl is replaced by a p-cresolyl group. It fully supports the turnover of methylmalonyl CoA mutase. The apparent K m values of methylmalonyl CoA mutase for Ado- PCC and AdoCbl are 354 and 64 nm, respectively [13]. A structural comparison of the protein–AdoCbl com- plexes for these two enzymes is shown in Fig. 1A,B. The glutamate mutase-bound nucleotide tail is located in a more crowded environment, where the space is more restricted. In particular, a bulkier residue, Leu59, is situated at the bottom of the nucleotide tail-binding pocket of glutamate mutase, but a small residue, Gly653, is located in the same position of methylmalo- nyl CoA mutase. The relatively restricted space in the nucleotide tail-binding pocket might account for the low activity and affinity of glutamate mutase towards AdoCbi-GDP. Our unpublished results also show that 0 0.02 0.04 0.06 0.08 0.1 0.12 A B C D 0 2 4 6 8 10 12 14 A AdoCbl (µM) 0 0.02 0.04 0.06 0.08 0.1 0 20 40 60 80 100 A MeCbi (µM) 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0 20 40 60 80 100 A AdoCbi (µM) 0 0.01 0.02 0.03 0.04 0.05 0.06 0 20 40 60 80 100 A AdoCbi-GDP (µM) Fig. 4. Binding of AdoCbl and its analogs to glutamate mutase by equilibrium dialysis. (A) AdoCbl, (B) MeCbi, (C) AdoCbi, and (D) AdoCbi-GDP. The proteins, 20 l M MutE and 100 l M MutS in 0.1 mL buffer (50 mM Tris ⁄ HCl, pH 8.5, 2 mM dithiothreitol), were dialyzed against 1 mL buffer containing 50 m M Tris ⁄ HCl, pH 8.5, 2 mM dithiothreitol and cofactors. The data obtained were fitted using KALEIDA GRAPH software. Adenosylcobalamin-dependent glutamate mutase H P. Chen et al. 5964 FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS AdoCbl-dependent lysine aminomutase binds AdoCbl with a K d of 18 ± 4 lm. Neither AdoCbi nor Ado- Cbi-GDP efficiently support the catalysis of AdoCbl- dependent l-lysine or d-ornithine aminomutase [14,15]. In short, the manipulation of coenzyme B 12 by methyl- malonyl CoA mutase is quite different to that by glu- tamate mutase, l-lysine and d-ornithine aminomutase. Two mechanisms, electronic effect and steric effect, have been postulated to explain the enzyme-accelerated cobalt–carbon cleavage of AdoCbl [3,16]. AdoCbi-GDP is bound by methylmalonyl CoA mutase in ‘base-off’ form, and is capable of supporting the enzyme’s cataly- sis, suggesting that the electronic effect plays a minor role in cleavage of the cobalt–carbon bond. However, as far as we know, no experimental results from the studies of coenzyme–protein interactions have previously been provided to support the steric effect to explain the cobalt–carbon cleavage mechanism. The binding energy for AdoCbl comes from inter- actions between proteins and the cofactor. From the viewpoint of coenzyme molecule itself, these interac- tions can be divided into three parts: the ribonucleo- tide tail, corrin ring ⁄ cobalt–histidine ligation, and the adenosyl group (Fig. 6). As shown in Table 2, the apparent K d values of glutamate mutase for MeCbi and AdoCbi are 6.0 ± 0.9 and 18 ± 3, respectively. As shown in Table 2, the binding energy difference between MeCbi and AdoCbi is approximately 2.5 kJÆmol )1 . This result suggests that, in the absence of the ribonucleotide tail of AdoCbl, the enzyme’s active site cannot correctly accommodate the coen- zyme analog AdoCbi. In accordance with this result, the histidine residue on the conserved cobalamin- binding motif can coordinate to the cobalt atom when MeCbi is used as the cofactor (Fig. 5A). How- ever, only approximately 60% of the glutamate mutase-bound AdoCbi is in the ‘base-off ⁄ His-on’ form (Fig. 5B). Although AdoCbi-GDP cannot effi- ciently support catalysis, its modified ribonucleotide tail helps the histidine residue coordinate to the cobalt atom (Fig. 5C). Previous studies have shown that glutamate mutase binds AdoCbl, methylcobal- amin (MeCbl) and cob(II)alamin with similar affinity [17]. These results indicate that the ribonucleotide tail of AdoCbl is important in coenzyme binding. We hereby propose that the role of the ribonucleo- tide tail of AdoCbl is to distort the adenosyl group to fit into the enzyme’s active site during the coen- zyme-binding process. However, recent spectroscopic studies have indicated that the Co–C bond of gluta- mate mutase-bound AdoCbl is not weakened within the enzyme active site [18,19]. The correlation between the distortion of the adenosyl group and cleavage of the cobalt–carbon bond is still not clear. Although the precise mechanism remains obscure, the results presented here do shed some light on the cobalt–carbon cleavage mechanism of B 12 . Experimental procedures Materials AdoCbl and methylcobalamin (MeCbl) were obtained from Sigma (St Louis, MO, USA). SP–Sepharose Fast Flow cat- ion-exchange gel medium was purchased from GE Health- care (Uppsala, Sweden). The production and purification of glutamate mutase from C. tetanomorphum have been 0 0.1 0.2 0.3 0.4 0.5 0.6 A B C 350 400 450 500 550 600 650 700 Free MeCbi Protein-bound MeCbi A Wavelength (nm) 0 0.2 0.4 0.6 0.8 1 350 400 450 500 550 600 650 700 Free AdoCbi Protein-bound AdoCbi A Wavelength (nm) 0 0.05 0.1 0.15 0.2 0.25 0.3 350 400 450 500 550 600 650 700 Free AdoCbi-GDP Protein-bound AdoCbi-GDP A Wavelength (nm) Fig. 5. UV–visible spectra of free and glutamate mutase-bound MeCbi (A), AdoCbi (B) and AdoCbi-GDP (C). H P. Chen et al. Adenosylcobalamin-dependent glutamate mutase FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS 5965 described previously [1]. All chemicals used were of analyti- cal grade or higher. Preparation of MeCbi and AdoCbi Because the cobalt–carbon bond of cobalamin is light- sensitive, the following procedure was carried out in a dark environment. The chemical synthesis of AdoCbi and MeCbi was slightly modified from that described previously [20]. For this reaction, 0.5 g of AdoCbl or MeCbl was used. The products, AdoCbi or MeCbi, were separated from the reaction mixture using a SP–Sepharose Fast Flow cation- exchange column (2.6 · 40 cm). The column was equili- brated in 10 mm potassium phosphate buffer, pH 7.0. AdoCbi or MeCbi were eluted with a 500 mL gradient from 0 to 0.5 m KCl. The flow rate was 3 mLÆmin )1 ; 4 mL frac- tions were collected. Fractions containing AdoCbi or MeCbi were pooled separately. The yield was approximately 30%. Chemo-enzymatic preparation of AdoCbi-GDP The cobU gene from Salmonella typhimurium ATCC 19585 has been successfully cloned and over-expressed in Escheri- chia coli [21]. CobU protein, in 50 mm Tris ⁄ HCl, pH 8.5, and other solutions used for the reaction were made anaerobic and equilibrated using alternate cycles of vacuum and hydrated argon gas for 15 min. The 1.5 mL reaction mixture containing 1.5 mm GTP, 1.5 mm MgCl 2 ,1mm b-mercaptoethanol, 10 lm CobU and 250 lm AdoCbi was buffered in 100 mm Tris ⁄ HCl, pH 8.5. Each solution was N N H N N Co +3 N N H Co +3 CH 3 N N N N H 2 N O OH OH H H H H OH N N H Co +3 OH N N N N H 2 N O OH OH H H H H Corrin ring and His ligation Nucleotide tail Adenosyl group AdoCbl MeCbi AdoCbi No contribution Distortion Free energy change contributed by: Fig. 6. Illustrations of the binding free energy change contributed by each fragment in coenzyme B 12 . Table 2. Comparison of the k cat ⁄ K m value, dissociation constants and binding free energies of various coenzyme analogs. The k cat ⁄ K m value for AdoCbl is calculated from the results in [1]. Coenzyme analogs Upper ligand of cobalt k cat ⁄ K m (s )1 ÆlM )1 ) K d (lM) DG (kJÆmol )1 ) AdoCbi Adenosyl group (4.3 ± 1.7) · 10 )4 18 ± 3 25.19 ± 0.39 MeCbi Methyl group N ⁄ A 6.0 ± 0.9 27.72 ± 0.35 AdoCbi-GDP Adenosyl group (7.4 ± 3.9) · 10 )5 14 ± 3 25.79 ± 0.50 AdoCbl Adenosyl group 1.12 ± 0.09 3.7 ± 0.5 28.83 ± 0.31 Adenosylcobalamin-dependent glutamate mutase H P. Chen et al. 5966 FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS injected separately into a rubber-sealed 2 mL vial that had been flushed with argon for 10 min prior to use. The reac- tion was incubated at room temperature overnight and was terminated by incubation at 95 °C for 10 min. AdoCbi-GDP was isolated from the reaction mixture by reverse-phase HPLC on a 5lm, 25 cm · 4.6 mm, Supelco AscentisÔ C 18 column (Bellefonte, PA, USA). The eluents used were as follows: eluent A, 100 mm potassium phos- phate buffer, pH 6.5; eluent B, 100 mm potassium phos- phate buffer, pH 8.0 containing 50% CH 3 CN. The flow rate was 1 mLÆmin )1 . The following profile was used for separation: 2 min isocratic development with 98% A; 5 min linear gradient from 98% A to 75% A; 15 min linear gradi- ent from 75% A to 65% A; 3 min linear gradient from 65% A to 0% A; 10 min isocratic development with 100% B. Both analogs, AdoCbi and AdoCbi-GDP, were charac- terized by ESI-MS. NMR spectroscopy NMR spectra of AdoCbi-GDP were recorded on a Bruker AVANCE 600 AV system (Bruker BioSpin GmbH; Rhein- stetten, Germany) at 25 °C. Approximately 2 mg of AdoCbi- GDP dissolved in 0.25 mL H 2 O containing 10% D 2 O was used for the NMR experiment. Two-dimensional homo- nuclear (TOCSY and ROESY) and heteronuclear (HMQC and HMBC) spectra of AdoCbi-GDP were collected for the chemical shift assignment. The ROESY spectra were obtained with mixing times of 50 and 150 ms, to classify the relative strengths of the observed NOEs. All spectra were pro- cessed and analyzed by using topspin 2.1 software (Bruker BioSpin GmbH; Rheinstetten, Germany). Measurement of the binding of coenzyme analogs to proteins The binding of coenzyme analogs to glutamate mutase was measured by equilibrium dialysis. About 100 lLof20lm E component and 100 lm S component were loaded into microdialysis tubes. The protein solutions were dialyzed against 1 mL of 50 mm Tris buffer, pH 8.5, in the presence of various concentrations of coenzyme B 12 or its analogs at 4 °C overnight. The absorbance was recorded at 522 nm using an Amersham Bioscience Ultrospec 2100 spectropho- tometer; a sample of the corresponding dialysis buffer was used to subtract the contribution of unbound coenzyme analogs from the absorbance of the enzyme. The kaleida graph program (Synergy Software, Reading, PA, USA) was used to fit data to estimate the dissociation constant. Protein UV–visible spectra To determine the coordination state of the cobalt atom of enzyme-bound coenzyme analogs, 100 lL of protein solu- tion containing 400 lm S component, 100 lm E compo- nent, and 50 or 100 lm coenzyme analog was dialyzed against 1 mL 50 mm Tris buffer, pH 8.5, at 4 °Cinthe dark overnight, by which time equilibrium had been reached. Spectra were recorded using an Amersham Bio- science Ultrospec 2100 Pro spectrophotometer (Uppsala, Sweden); a sample of the dialysis buffer was used to sub- tract the contribution of unbound coenzyme analog from the spectra of the holoenzymes. Enzyme assay An HPLC-based method was used to assay glutamate mutase activity [22]. The assay was made irreversible by coupling the formation of 3-methylaspartate to the pro- duction of mesaconate through deamination by methylas- partase. In a typical reaction, 10 lm E component and 50 lm S component proteins were used in a total volume of 100 lL containing 2 mm MgCl 2 ,40mml-glutamate and 50 mm Tris buffer, pH 8.5. The K m and k cat for Ado- Cbi were determined in the presence of 10, 25, 50, 75 and 120 lm cofactor, and the K m and k cat for AdoCbi-GDP were determined in the presence of 20, 70, 100, 150 and 200 lm cofactor. The reaction was initiated by adding l-glutamate and incubating at room temperature for 15 min. The formation of mesaconate was then analyzed by reverse-phase HPLC on a C 18 column (4.6 · 250 mm) as described previously [22]. Acknowledgements This work was supported by grants NSC-94-2320-B- 027-002 and NSC-95-2113-M-027-005-MY2 from the National Scientific Council, Taiwan, Republic of China, to H P.C. References 1 Holloway DE & Marsh ENG (1994) Adenosylcobala- min-dependent glutamate mutase from Clostridium tetanomorphum. 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Protein Expr Purif 42, 178–181. 22 Marsh ENG (1995) Tritium isotope effects in adenosyl- cobalamin-dependent glutamate mutase: implications for the mechanism. Biochemistry 34, 7542–7547. Adenosylcobalamin-dependent glutamate mutase H P. Chen et al. 5968 FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS . Interactions between coenzyme B 12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum Hao-Ping. results of coenzyme- binding and kinetic studies of AdoCbl analogs with glutamate mutase. Results Synthesis of MeCbi, AdoCbi and AdoCbi-GDP MeCbi and AdoCbi