Báo cáo khoa học: Interactions between coenzyme B12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum pot

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Interactions between coenzyme B12analogs andadenosylcobalamin-dependent glutamate mutasefrom Clostridium tetanomorphumHao-Ping Chen1, Huei-Ju Hsu1, Fang-Ciao Hsu1, Chien-Chen Lai2and Chung-Hua Hsu31 Institute of Biotechnology, National Taipei University of Technology, Taiwan2 Institute of Molecular Biology, National Chung-Hsing University, Taichung, Taiwan3 Department of Agricultural Chemistry, National Taiwan University, Taipei, TaiwanGlutamate mutase from Clostridium tetanomorphum isone of a group of adenosylcobalamin (AdoCbl)-depen-dent mutases that catalyzes the inter-conversion ofl-glutamate and threo-b-methyl-l-aspartate. It com-prises two weakly-associating subunits, MutS andMutE, which combine with AdoCbl to form the activeholo-enzyme [1]. The coenzyme is known to be boundby glutamate mutase in ‘base-off ⁄ His-on’ mode [2]. Asshown in Fig. 1A, the lower axial ligand of the cobaltatom, 5,6-dimethylbenzimidazole, is replaced by a his-tidine residue within a conserved B12-binding motif,DXHXXG(14–19). Model studies have shown that thecobalt–carbon bond dissociation energy of the cofactoris sensitive to changes in the pKaof the lower axialbase [3]. This has led to speculation that proteinsmight modulate the pKaof the histidine via thehydrogen bond between the His–Asp pair and so ‘finetune’ the reactivity of AdoCbl. Mutations of eitherresidue result in significant impairment of the protein’scoenzyme-binding ability, as well as its catalyticability [4].The biosynthesis of AdoCbl is a very complicatedprocess. 5¢-deoxyadenosyl- cobinamide (AdoCbi) andAdoCbi-GDP are intermediates during the biosynthesisof AdoCbl (Fig. 2A). Previous studies have shown thatAdoCbl-dependent methylmalonyl CoA mutase bindsboth coenzyme analogs in ‘base-off’ mode, which indi-cates that the histidine residue located on the conservedcobalamin-binding motif is unable to coordinate to thecobalt atom [5,6]. However, the AdoCbi-GDP-reconsti-Keywordsadenosylcobalamin; adenosylcobinamide;AdoCbi-GDP; B12; glutamate mutaseCorrespondenceH P. Chen, Institute of Biotechnology,National Taipei University of Technology 1,Sec 3, Chung-Hsiao East Road, Taipei 106,TaiwanFax: +886 2 27317117Tel: +886 2 27712171 ext. 2528E-mail: hpchen@ntut.edu.tw(Received 14 August 2008, revised 30September 2008, accepted 2 October2008)doi:10.1111/j.1742-4658.2008.06724.xAdenosylcobalamin (AdoCbl)-dependent glutamate mutase from Clostrid-ium tetanomorphum comprises two weakly-associating subunits, MutS andMutE, which combine with AdoCbl to form the active holo-enzyme. Threecoenzyme analogs, methylcobinamide (MeCbi), adenosylcobinamide (Ado-Cbi) and adeosylcobinamide-GDP (AdoCbi-GDP), were synthesized atmilligram scale. Equilibrium dialysis was used to measure the binding ofcoenzyme B12analogs to glutamate mutase. Our results show that, unlikeAdoCbl-dependent methylmalonyl CoA mutase, the ratio kcat⁄ Kmdecreased approximately 104-fold in both cases when AdoCbi or AdoCbi-GDP was used as the cofactor. The coenzyme analog-binding studies showthat, in the absence of the ribonucleotide tail of AdoCbl, the enzyme’sactive site cannot correctly accommodate the coenzyme analog AdoCbi.The results presented here shed some light on the cobalt–carbon cleavagemechanism of B12.AbbreviationsAdoCbi, 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 FEBStuted enzyme is catalytically active. More importantly,the kcat⁄ Kmof methylmalonyl CoA mutase is onlyfour-fold lower when AdoCbi-GDP is used as cofactor[5,6]. This unexpected result suggests that coordinationby the lower axial ligand is not essential in the case ofmethylmalonyl CoA mutase. To study the reactivity ofglutamate mutase toward these coenzyme analogs, achemo-enzymatic method was developed to synthesizeAdoCbi-GDP at the milligram scale. Our results showthat, in contrast to methylmalonyl CoA mutase, neitherAdoCbi nor AdoCbi-GDP can efficiently act as cofac-tor for glutamate mutase [5]. The binding of AdoCbland three coenzyme analogs, methylcobinamide (MeC-bi), AdoCbi and AdoCbi-GDP, to glutamate mutasewas measured by equilibrium dialysis. Kinetic proper-ties towards AdoCbi and AdoCbi-GDP were alsoinvestigated. Here, we report the results of coenzyme-binding and kinetic studies of AdoCbl analogs withglutamate mutase.ResultsSynthesis of MeCbi, AdoCbi and AdoCbi-GDPMeCbi and AdoCbi were successfully separated fromunreacted MeCbl and AdoCbl and the dealkylated sideproducts using an SP–Sepharose ion-exchange column.The relative molecular masses of MeCbi and AdoCbidetermined by ESI-MS were 1004.5 and 1240, whichcompare favorably with calculated relative molecularmasses for MeCbi and AdoCbi of 1004.1 and 1239.6,respectively. The bifunctional enzyme CobU (adenosyl-cobinamide kinase ⁄ adenosyl-cobinamide-phosphateguanylyltransferase) is involved in biosynthesis andassembly of the nucleotide loop of cobalamin [7,8](Fig. 2A,B) Using chemically synthesized AdoCbi as theCobU substrate, AdoCbi-GDP was enzymatically pre-pared in large quantities. The yield of AdoCbi-GDPcould be significantly enhanced by using phenol ⁄ dichlo-romethane extraction to remove the salt component ofthe AdoCbi solution. The recovery of AdoCbi-GDP byreverse-phase HPLC was very reproducible (Fig. 3). Therelative molecular mass of AdoCbi-GDP determined byESI-MS was 1664.4, and the calculated relative molecu-lar mass of AdoCbi-GDP is 1664.6. The HPLC methodthat we developed in this study is quite straightforward,separating AdoCbi and AdoCbi-GDP directly withoutfurther modification. In contrast, the reactant and prod-uct, AdoCbi and AdoCbi-GDP, were analyzed in theform of (CN)2Cbi and (CN)2Cbi-GDP, respectively, inprevious reports [7,8].1H-NMR spectra for MeCbi andAdoCbi have been published previously [9,10]. The600 MHz NMR spectrum of AdoCbi-GDP inD2O ⁄ H2O was analyzed using two-dimensional COSYand NOESY experiments. The results are summarizedin Table 1 and Fig. 2B.D14C15H16G120T121S61V60L59G92G91Y117I22L23A118I334E330T94R66A67G68E subunit(53.7 kDa)S subuniT(14 kDa)H610D608G609G686G685G613G653V654S655Y705T709T706I617E370E247Y243Y89Q330L374ABFig. 1. (A) Model of glutamate mutase showing AdoCbl bound between the MutS and MutE subunits. The coenzyme-binding domain is onthe 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 mutaseFEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS 5961ONH2CONH2CONH2H2NOCH2NOCCONH2OOHNHHNNNNNH2OOHHOHHCoNNNNAdoCbiONH2CONH2CONH2H2NOCH2NOCCONH2OO-POHNHOONNNNNH2OOHHOHHCoNNNNNNNNNH2OOHOO-POOAdoCbi-GDPONH2CONH2CONH2H2NOCH2NOCCONH2ONHH3CHNNNNNH2OOHHOHHCoNNNNAdoCbi-PPOOO-OATPADPGTPPPiCobinamidekinaseCobinamidekinaseONH2CONH2CONH2H2NOCH2NOCCONH2ONNOOHHOOPONHHOO-NNNNNH2OOHHOHHCoNNNNAdoCblα-ribazoleGMPCobalaminsynthaseAPrAdoCbi-GDPNNNNCoNH2OH2NH2NONH2OONH2ONH2ONHOPOPOOONOHNNNHONH2R5463217891011121314151617181920252627303132353736384142464748495053545556576061Pr1Pr23R2R3R4R5R=NNNNOOHOHH2CNH2A15A14A13A12A11O-O-OOR1A2A8abcd43efgB2A4A5BFig. 2. (A) Schematic representation of thefinal steps of the de novo AdoCbl biosyn-thetic pathway. (B) The chemical structureof 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 FEBSDetermination of dissociation constants forcofactors by equilibrium dialysisThe binding of AdoCbl, MeCbi, AdoCbi and AdoCbi-GDP to glutamate mutase was investigated by equilib-rium dialysis. Figure 4 shows the analog bindingcurves with a fixed concentration of glutamate mutase.AdoCbl, MeCbi, AdoCbi and AdoCbi-GDP werebound with apparent Kdvalues 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 complexesThe UV–visible spectra of cobalamins provide auseful tool to examine the coordination state ofcobalt. The UV–visible absorption spectra of theMeCbi-glutamate mutase, AdoCbi-glutamate mutaseand AdoCbi-GDP-glutamate mutase complexes weremeasured. A red shift was observed in the spectra ofprotein-bound MeCbi, AdoCbi and AdoCbi-GDP.The 522 nm absorption maximum suggests that thehistidine residue occupies the lower axial ligand posi-tion of the cobalt atom. However, we estimate thatapproximately 55–60% of the AdoCbi–glutamatemutase 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-GDP300 200 100 0 300 200 100 0 0 5 10 15 20 Time 25 30 35 40 45 Fig. 3. Purification of AdoCbi-GDP from the reaction mixtureby reverse-phase HPLC. (A) Before the CobU enzymatic reaction.(B) After the CobU enzymatic reaction.Table 1. 600 MHz1H-NMR data for AdoCbi-GDP. d, doublet; q,quadruplet; s, singlet; t, triplet; td, triplet of doublets; dd, doublet ofdoublets.AssignmentSignaltypeChemical shiftsAdoCbi-GDP(pH 7.0, 25 °C)(p.p.m.)J couplings(AdoCbi-GDP)(Hz)CorrinmethylC20 s 0.77C25 s 1.38C35 s 2.38C36 s 1.79C46 s 0.83C47 s 1.57C53 s 2.36C54 s 1.12Corrin CH C3 m 4.19C8 m 3.76C10 s 6.92C13 dd 3.35 5.77, 3.23C18 td 2.8 10.4, 3.58C19 d 4.62 3.58Corrin CH2side chainC26 d 2.62, 2.3 14.6C30 m 1.96, 1.85C31 m 2.44C37 d 2.22, 1.74 14.8C41 m 1.93, 1.81C42 m 2.32, 2.24C48 m 1.92, 1.77C49 m 2.16C55 m 1.75C56 m 2.27C60 d 2.63, 2.40 10.4Aminopropan-2-ol side chainPr1(CH2) t 3.30, 3.185 5.5Pr2(CH) m 4.38Pr3(CH3) d 1.21 6.5Loop ribose R1 d 5.85 6.81R2 m 4.68R3 dd 4.46 4.87, 3.47R4 m 4.27R5 m 4.15Adenosyl A2 s 8.20A8 s 8.02A11 d 5.60 3.54A12 dd 4.40 5.54, 5.75A13 dd 3.75 6.54, 5.75A14 dd 1.91 9.3, 6.54A15 d, dd 0.50, 0.32 8.6;8.6, 9.3Base B2 s 8.01NH s 8.20s 7.94s 7.84s 7.6s 7.57s 7.3s 7.27s 7.09s 7.02s 6.88s 6.86s 6.60s 6.57s 6.36H P. Chen et al. Adenosylcobalamin-dependent glutamate mutaseFEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS 5963Enzyme assayIn order to investigate the role of the ribonucleotide tailof AdoCbl in catalysis, the coenzyme analogs were usedto examine the enzymatic activity. Our results indicatethat, perhaps not surprisingly, MeCbi is a totally inac-tive coenzyme. The Kmvalues for AdoCbi and Ado-Cbi-GDP were 26 ± 8 and 75 ± 28 lm, respectively,and the kcatvalues were (9.8 ± 1.0) · 10)3Æs)1and(4.5 ± 0.8) · 10)3Æs)1, respectively. In both cases, thekcat⁄ Kmwas decreased by approximately 104-foldcompared with that of AdoCbl.DiscussionBoth methylmalonyl CoA mutase and glutamatemutase belong to the subfamily of B12-dependent car-bon-skeleton mutases, but their 1,2-rearrangementmechanisms are obviously different [11]. Previousstudies have shown that (a) AdoCbi does not supportthe turnover of methylmalonyl CoA mutase, but Ado-Cbi-GDP does, and (b) the enzyme binds both AdoCbiand AdoCbi-GDP in ‘base-off ⁄ His-off’ mode. Theresults presented here indicate that, in contrast tomethylmalonyl CoA mutase, the kcat⁄ Kmof glutamatemutase for both analogs decreased by approximately104-fold. These results suggest that the ribonucleotidetail of AdoCbl plays an important role in catalysis inthe case of glutamate mutase. In addition, both cofac-tor analogs tested are bound by glutamate mutase in‘base-off ⁄ His-on’ mode. Histidine–cobalt ligationtherefore cannot efficiently facilitate turnover of theenzyme in the absence of the ribonucleotide tail ofAdoCbl. It is apparent that glutamate mutase is mech-anistically different from methylmalonyl CoA mutase.Significant differences in the affinity for AdoCblbetween these two enzymes appear to exit. Methylmal-onyl CoA mutase binds AdoCbl very tightly with a Kdof 0.17 lm, while glutamate mutase binds AdoCblrelatively weakly with a Kdbetween 1.8 and 6.8 lm[1]. Moreover, glutamate mutase is very sensitive toperturbation of the cofactor’s nucleotide tail, whilemethylmalonyl CoA mutase is not. (Benzimidazolribo-furanosyl)-adenosylcobinamide [(Bza)AdoCba] is acoenzyme B12analog in which the dimethylbenzimi-dazole moiety of AdoCbl is replaced by benzimidazole.Previous studies have shown that the apparent Kmofglutamate mutase for (Bza)AdoCba is 0.5 lm, whilethat for AdoCbl is 18 lm under similar conditions[12]. However, the only difference between AdoCbland (Bza)AdoCba is two methyl groups. In contrast,(Co-b-5¢-Deoxyadenosin-5¢-yl)-(p-cresyl)cobamide (Ado-PCC) is another ‘base-off’ coenzyme B12analog inwhich the dimethylbenzimidazole moiety of AdoCbl isreplaced by a p-cresolyl group. It fully supports theturnover of methylmalonyl CoA mutase. The apparentKmvalues 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 locatedin a more crowded environment, where the space ismore restricted. In particular, a bulkier residue, Leu59,is situated at the bottom of the nucleotide tail-bindingpocket of glutamate mutase, but a small residue,Gly653, is located in the same position of methylmalo-nyl CoA mutase. The relatively restricted space in thenucleotide tail-binding pocket might account for thelow activity and affinity of glutamate mutase towardsAdoCbi-GDP. Our unpublished results also show that0 0.02 0.04 0.06 0.08 0.1 0.12 A B C D 0 2 4 6 8 10 12 14 AAdoCbl (µM) 0 0.02 0.04 0.06 0.08 0.1 0 20 40 60 80 100AMeCbi (µM) 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0 20 40 60 80 100 AAdoCbi (µM) 0 0.01 0.02 0.03 0.04 0.05 0.06 0 20 40 60 80 100AAdoCbi-GDP (µM) Fig. 4. Binding of AdoCbl and its analogs toglutamate mutase by equilibrium dialysis.(A) AdoCbl, (B) MeCbi, (C) AdoCbi, and (D)AdoCbi-GDP. The proteins, 20 lM MutE and100 lM MutS in 0.1 mL buffer (50 mMTris ⁄ HCl, pH 8.5, 2 mM dithiothreitol), weredialyzed against 1 mL buffer containing50 mM Tris ⁄ HCl, pH 8.5, 2 mM dithiothreitoland cofactors. The data obtained were fittedusingKALEIDA 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 FEBSAdoCbl-dependent lysine aminomutase binds AdoCblwith a Kdof 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 B12by 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-acceleratedcobalt–carbon cleavage of AdoCbl [3,16]. AdoCbi-GDPis 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 minorrole in cleavage of the cobalt–carbon bond. However, asfar as we know, no experimental results from the studiesof coenzyme–protein interactions have previously beenprovided to support the steric effect to explain thecobalt–carbon cleavage mechanism.The binding energy for AdoCbl comes from inter-actions between proteins and the cofactor. From theviewpoint of coenzyme molecule itself, these interac-tions can be divided into three parts: the ribonucleo-tide tail, corrin ring ⁄ cobalt–histidine ligation, and theadenosyl group (Fig. 6). As shown in Table 2, theapparent Kdvalues of glutamate mutase for MeCbiand AdoCbi are 6.0 ± 0.9 and 18 ± 3, respectively.As shown in Table 2, the binding energy differencebetween MeCbi and AdoCbi is approximately2.5 kJÆmol)1. This result suggests that, in the absenceof the ribonucleotide tail of AdoCbl, the enzyme’sactive 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 atomwhen MeCbi is used as the cofactor (Fig. 5A). How-ever, only approximately 60% of the glutamatemutase-bound AdoCbi is in the ‘base-off ⁄ His-on’form (Fig. 5B). Although AdoCbi-GDP cannot effi-ciently support catalysis, its modified ribonucleotidetail helps the histidine residue coordinate to thecobalt atom (Fig. 5C). Previous studies have shownthat glutamate mutase binds AdoCbl, methylcobal-amin (MeCbl) and cob(II)alamin with similar affinity[17]. These results indicate that the ribonucleotidetail 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 groupto fit into the enzyme’s active site during the coen-zyme-binding process. However, recent spectroscopicstudies have indicated that the Co–C bond of gluta-mate mutase-bound AdoCbl is not weakened withinthe enzyme active site [18,19]. The correlationbetween the distortion of the adenosyl group andcleavage of the cobalt–carbon bond is still not clear.Although the precise mechanism remains obscure,the results presented here do shed some light on thecobalt–carbon cleavage mechanism of B12.Experimental proceduresMaterialsAdoCbl and methylcobalamin (MeCbl) were obtained fromSigma (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 ofglutamate mutase from C. tetanomorphum have been0 0.1 0.2 0.3 0.4 0.5 0.6 A B C 350 400 450 500 550 600 650 700 Free MeCbiProtein-bound MeCbiAWavelength (nm)0 0.2 0.4 0.6 0.8 1 350 400 450 500 550 600 650 700 Free AdoCbiProtein-bound AdoCbiAWavelength (nm)0 0.05 0.1 0.15 0.2 0.25 0.3 350 400 450 500 550 600 650 700 Free AdoCbi-GDPProtein-bound AdoCbi-GDPAWavelength (nm)Fig. 5. UV–visible spectra of free and glutamate mutase-boundMeCbi (A), AdoCbi (B) and AdoCbi-GDP (C).H P. Chen et al. Adenosylcobalamin-dependent glutamate mutaseFEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS 5965described previously [1]. All chemicals used were of analyti-cal grade or higher.Preparation of MeCbi and AdoCbiBecause the cobalt–carbon bond of cobalamin is light-sensitive, the following procedure was carried out in a darkenvironment. The chemical synthesis of AdoCbi and MeCbiwas slightly modified from that described previously [20].For this reaction, 0.5 g of AdoCbl or MeCbl was used. Theproducts, AdoCbi or MeCbi, were separated from thereaction 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 from0 to 0.5 m KCl. The flow rate was 3 mLÆmin)1; 4 mL frac-tions were collected. Fractions containing AdoCbi or MeCbiwere pooled separately. The yield was approximately 30%.Chemo-enzymatic preparation of AdoCbi-GDPThe cobU gene from Salmonella typhimurium ATCC 19585has 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 madeanaerobic and equilibrated using alternate cycles of vacuumand hydrated argon gas for 15 min. The 1.5 mL reactionmixture containing 1.5 mm GTP, 1.5 mm MgCl2,1mmb-mercaptoethanol, 10 lm CobU and 250 lm AdoCbi wasbuffered in 100 mm Tris ⁄ HCl, pH 8.5. Each solution wasNNHNNCo+3NNHCo+3CH3NNNNH2NOOHOHHHHHOHNNHCo+3OHNNNNH2NOOHOHHHHHCorrin ring and His ligationNucleotide tailAdenosyl groupAdoCblMeCbi AdoCbiNo contributionDistortionFree energy changecontributed by:Fig. 6. Illustrations of the binding free energy change contributed by each fragment in coenzyme B12.Table 2. Comparison of the kcat⁄ Kmvalue, dissociation constants and binding free energies of various coenzyme analogs. The kcat⁄ Kmvaluefor AdoCbl is calculated from the results in [1].Coenzyme analogs Upper ligand of cobalt kcat⁄ Km(s)1ÆlM)1) Kd(lM) DG (kJÆmol)1)AdoCbi Adenosyl group (4.3 ± 1.7) · 10)418 ± 3 25.19 ± 0.39MeCbi Methyl group N ⁄ A 6.0 ± 0.9 27.72 ± 0.35AdoCbi-GDP Adenosyl group (7.4 ± 3.9) · 10)514 ± 3 25.79 ± 0.50AdoCbl Adenosyl group 1.12 ± 0.09 3.7 ± 0.5 28.83 ± 0.31Adenosylcobalamin-dependent glutamate mutase H P. Chen et al.5966 FEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBSinjected separately into a rubber-sealed 2 mL vial that hadbeen flushed with argon for 10 min prior to use. The reac-tion was incubated at room temperature overnight and wasterminated by incubation at 95 °C for 10 min.AdoCbi-GDP was isolated from the reaction mixture byreverse-phase HPLC on a 5lm, 25 cm · 4.6 mm, SupelcoAscentisÔ C18column (Bellefonte, PA, USA). The eluentsused 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% CH3CN. The flowrate was 1 mLÆmin)1. The following profile was used forseparation: 2 min isocratic development with 98% A; 5 minlinear gradient from 98% A to 75% A; 15 min linear gradi-ent from 75% A to 65% A; 3 min linear gradient from65% A to 0% A; 10 min isocratic development with 100%B. Both analogs, AdoCbi and AdoCbi-GDP, were charac-terized by ESI-MS.NMR spectroscopyNMR spectra of AdoCbi-GDP were recorded on a BrukerAVANCE 600 AV system (Bruker BioSpin GmbH; Rhein-stetten, Germany) at 25 °C. Approximately 2 mg of AdoCbi-GDP dissolved in 0.25 mL H2O containing 10% D2O wasused for the NMR experiment. Two-dimensional homo-nuclear (TOCSY and ROESY) and heteronuclear (HMQCand HMBC) spectra of AdoCbi-GDP were collected for thechemical shift assignment. The ROESY spectra wereobtained with mixing times of 50 and 150 ms, to classify therelative strengths of the observed NOEs. All spectra were pro-cessed and analyzed by using topspin 2.1 software (BrukerBioSpin GmbH; Rheinstetten, Germany).Measurement of the binding of coenzymeanalogs to proteinsThe binding of coenzyme analogs to glutamate mutase wasmeasured by equilibrium dialysis. About 100 lLof20lmE component and 100 lm S component were loaded intomicrodialysis tubes. The protein solutions were dialyzedagainst 1 mL of 50 mm Tris buffer, pH 8.5, in the presenceof various concentrations of coenzyme B12or its analogs at4 °C overnight. The absorbance was recorded at 522 nmusing an Amersham Bioscience Ultrospec 2100 spectropho-tometer; a sample of the corresponding dialysis buffer wasused to subtract the contribution of unbound coenzymeanalogs from the absorbance of the enzyme. The kaleidagraph program (Synergy Software, Reading, PA, USA)was used to fit data to estimate the dissociation constant.Protein UV–visible spectraTo determine the coordination state of the cobalt atom ofenzyme-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 dialyzedagainst 1 mL 50 mm Tris buffer, pH 8.5, at 4 °Cinthedark overnight, by which time equilibrium had beenreached. 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 fromthe spectra of the holoenzymes.Enzyme assayAn HPLC-based method was used to assay glutamatemutase activity [22]. The assay was made irreversible bycoupling the formation of 3-methylaspartate to the pro-duction of mesaconate through deamination by methylas-partase. In a typical reaction, 10 lm E component and50 lm S component proteins were used in a total volumeof 100 lL containing 2 mm MgCl2,40mml-glutamateand 50 mm Tris buffer, pH 8.5. The Kmand kcatfor Ado-Cbi were determined in the presence of 10, 25, 50, 75 and120 lm cofactor, and the Kmand kcatfor AdoCbi-GDPwere determined in the presence of 20, 70, 100, 150 and200 lm cofactor. The reaction was initiated by addingl-glutamate and incubating at room temperature for15 min. The formation of mesaconate was then analyzedby reverse-phase HPLC on a C18column (4.6 · 250 mm)as described previously [22].AcknowledgementsThis work was supported by grants NSC-94-2320-B-027-002 and NSC-95-2113-M-027-005-MY2 from theNational Scientific Council, Taiwan, Republic ofChina, to H P.C.References1 Holloway DE & Marsh ENG (1994) Adenosylcobala-min-dependent glutamate mutase from Clostridiumtetanomorphum. J Biol Chem 269, 20425–20430.2 Zelder O, Beatrix B, Kroll F & Buckel W (1995)Coordination of a histidine residue of the protein-com-ponent S to the cobalt atom in coenzyme B12-dependentglutamate mutase from Clostridium cochlearium. FEBSLett 369, 252–254.3 Halpern J (1985) Mechanisms of coenzyme B12-depen-dent rearrangements. Science 227, 869–875.4 Chen HP & Marsh ENG (1997) How enzymes controlthe reactivity of adenosylcobalamin: effect on coenzymebinding and catalysis of mutations in the conserved his-tidine-aspartate pair of glutamate mutase. Biochemistry36, 7884–7889.H P. Chen et al. Adenosylcobalamin-dependent glutamate mutaseFEBS Journal 275 (2008) 5960–5968 ª 2008 The Authors Journal compilation ª 2008 FEBS 59675 Chowdhury S & Banerjee R (1999) Role of the dimeth-ylbenzimidazole tail in the reaction catalyzed by coen-zyme B12-dependent methylmalonyl-CoA mutase.Biochemistry 38, 15287–15294.6 Chowdhury S, Thomas MG, Escalante-Semerena JC &Banerjee R (2001) The coenzyme B12analog 5’-deoxy-adenosylcobinamide-GDP supports catalysis by methyl-malonyl-CoA mutase in the absence of trans-ligandcoordination. J Biol Chem 276 , 1015–1019.7 O’Toole GA & Escalante-Semerena JC (1995) Purifica-tion and characterization of the bifunctional CobUenzyme of Salmonella typhimurium LT2. Evidence for aCobU-GMP intermediate. J Biol Chem 270, 23560–23569.8 Thomas MG, Thompson TB, Rayment I & Escalante-Semerena JC (2000) Analysis of the adenosylcobinamidekinase ⁄ adenosylcobinamide-phosphate guanylyltransfer-ase (CobU) enzyme of Salmonella typhimurium LT2.Identification of residue His-46 as the site of guanylyla-tion. J Biol Chem 275, 27576–27586.9 Brown KL, Zou X & Salmin L (1991) Facile a ⁄ b dia-stereomerism in organocobalt corrins. Generality of thephenomenon and characterization of additional a-dia-stereomers. Inorg Chem 30, 1949–1953.10 Pagano TG, Yohannes PG, Hay BP, Scott JR, FinkeRG & Marzilli LG (1989) Solution behavior and com-plete proton and carbon-13 NMR assignments of thecoenzyme B12derivative (5’-deoxyadenosyl)cobinamideusing modern 2D NMR experiments, including600 MHz proton NMR data. J Am Chem Soc 111,1484–1491.11 Banerjee R & Rasdale SW (2003) The many faces ofvitamin B12: catalysis by cobalamin-dependentenzymes. Annu Rev Biochem 72, 209–247.12 Holloway DE, Harding SE & Marsh ENG (1996)Adenosylcobalamin-dependent glutamate mutase:properties of a fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit.Biochem J 320, 825–830.13 Poppe L, Stupperich E, Hull WE, Buckel T & Retey J(1997) A base-off analogue of coenzyme-B12with amodified nucleotide loop1H-NMR structure analysisand kinetic studies with (R)-methylmalonyl-CoAmutase, glycerol dehydratase, and diol dehydratase. EurJ Biochem 250, 303–307.14 Chang CH & Frey PA (2000) Cloning, sequencing,heterologous expression, purification, and characteriza-tion of adenosylcobalamin-dependent d-lysine 5,6-ami-nomutase from Clostridium sticklandii. J Biol Chem 275,106–114.15 Chen HP, Wu SH, Lin YL, Chen CM & Tsay SS(2001) Cloning, sequencing, heterologous expression,purification and characterization of adenosylcobalamin-dependent d-ornithine aminomutase from Clostridiumsticklandii. J Biol Chem 276, 44744–44750.16 Pratt JM (1985) The B12-dependent isomerase enzymes;how the protein controls the active site. Chem Soc Rev14, 161–170.17 Chen HP & Marsh ENG (1997) Adenosylcobalamin-dependent glutamate mutase: examination of substrateand coenzyme binding in an engineered fusion proteinpossessing simplified subunit structure and kinetic prop-erties. Biochemistry 36, 14939–14945.18 Brooks AJ, Fox CC, Marsh ENG, Vlasie M, BanerjeeR & Brunold TC (2005) Electronic structure studies ofthe adenosylcobalamin cofactor in glutamate mutase.Biochemistry 44, 15167–15181.19 Sension RJ, Cole AG, Harris AD, Fox CC, WoodburyNW, Lin S & Marsh ENG (2004) Photolysis andrecombination of adenosylcobalamin bound toglutamate mutase. J Am Chem Soc 126, 1598–1599.20 Hay BP & Finke RG (1987) Thermolysis of the Co–Cbond in adenosylcorrins. 3. Quantification of the axialbase effecting adenosylcobalamin by the synthesis andthermolysis of axial base-free adenosylcobinamide.Insights into the energetics of enzyme-assisted cobalt–carbon bond homolysis. J Am Chem Soc 109, 8012–8018.21 Hsu FC, Ho TJ, Lai CC, Lin CF & Chen HP (2005)Cloning, sequencing, expression, and single-step purifi-cation of the adenosylcobinamide kinase ⁄ adenosylcobi-namide-phosphate guanylyltransferase (CobU) fromSalmonella typhimurium ATCC 19585. Protein ExprPurif 42, 178–181.22 Marsh ENG (1995) Tritium isotope effects in adenosyl-cobalamin-dependent glutamate mutase: implicationsfor 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 B12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum Hao-Ping. results of coenzyme- binding and kinetic studies of AdoCbl analogs with glutamate mutase. ResultsSynthesis of MeCbi, AdoCbi and AdoCbi-GDPMeCbi and AdoCbi
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Xem thêm: Báo cáo khoa học: Interactions between coenzyme B12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum pot, Báo cáo khoa học: Interactions between coenzyme B12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum pot, Báo cáo khoa học: Interactions between coenzyme B12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum pot