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A New Mode of B Binding and the Direct Participation of A Research Article 997 A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase Naoki Shibata1, Jun Masuda1, Takamasa Tobimatsu2, Tetsuo Toraya2*, Kyoko Suto1, Yukio Morimoto1 and Noritake Yasuoka1* Background: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin Addresses: 1Department of Life Science, Himeji (coenzyme B ) dependent conversion of 1,2-diols to the corresponding Institute of Technology, 1475-2 Kanaji, Kamigori, 12 Ako-gun, Hyogo 678-1297, Japan and 2Department aldehydes. The reaction initiated by homolytic cleavage of the cobalt–carbon bond of Bioscience and Biotechnology, Faculty of α β γ of the coenzyme proceeds by a radical mechanism. The enzyme is an 2 2 2 Engineering, Okayama University, Tsushima-Naka, heterooligomer and has an absolute requirement for a potassium ion for catalytic Okayama 700-8530, Japan. activity. The crystal structure analysis of a diol dehydratase–cyanocobalamin *Corresponding authors. complex was carried out in order to help understand the mechanism of action of E-mail: [email protected] this enzyme. [email protected] Results: The three-dimensional structure of diol dehydratase in complex with Key words: B12 enzyme, diol dehydratase, radicals, cyanocobalamin was determined at 2.2 Å resolution. The enzyme exists as a reaction mechanism, TIM barrel αβγ dimer of heterotrimers ( )2. The cobalamin molecule is bound between the Received: 16 March 1999 α and β subunits in the ‘base-on’ mode, that is, 5,6-dimethylbenzimidazole of Revisions requested: 7 April 1999 the nucleotide moiety coordinates to the cobalt atom in the lower axial position. Revisions received: 21 April 1999 α β α Accepted: 27 April 1999 The subunit includes a ( / )8 barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl Published: 28 July 1999 groups of the substrate coordinate directly to the potassium ion. Structure August 1999, 7:997–1008 Conclusions: This is the first crystallographic indication of the ‘base-on’ mode of http://biomednet.com/elecref/0969212600700997 cobalamin binding. An unusually long cobalt–base bond seems to favor homolytic © Elsevier Science Ltd ISSN 0969-2126 cleavage of the cobalt–carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested. Introduction protected by enzymes from undesired side reactions? Vitamin B12 or cobalamin is taken up by the cells and con- Crystal structures of cobalamin enzymes would give the verted into two coenzyme forms: adenosylcobalamin most important clue for solving these issues. X-ray crystallo- (coenzyme B12; Figure 1a) and methylcobalamin. The bio- graphic studies by Drennan et al. [7] on a cobalamin- chemical roles of these two coenzymes are different — the binding fragment of Escherichia coli methionine synthase former serves as a prosthetic group for the enzymes cat- and by Mancia et al. [8,9] on the whole enzyme of Propi- alyzing carbon-skeleton rearrangements, amino group onibacterium shermanii methylmalonyl-CoA mutase, migration, elimination, and ribonucleotide reduction, and together with electron paramagnetic resonance (EPR) the latter serves as a cofactor for the enzymes that catalyze studies on these enzymes [7,10] and Clostridium cochlear- methyl transfer reactions. The mechanisms of action of ium glutamate mutase [11], revealed that cobalamin is these coenzymes are also quite different. Adenosylcobal- bound to these enzymes in the so-called ‘base-off’ form amin-dependent enzymatic reactions are initiated by with histidine ligation — that is, the 5,6-dimethylbenzim- homolysis of the Co–C bond of the enzyme-bound cofac- idazole ligand is displaced from the cobalt and replaced tor and catalyzed by a radical mechanism [1–5] by the imidazole group of a histidine residue of the pro- (Figure 1b). In contrast, methyl-transfer reactions cat- teins in the lower axial position. Coordination of a histi- alyzed by methylcobalamin-dependent enzymes proceed dine residue to the cobalt atom of p-cresolylcobamide was by an ionic mechanism [6]. found with a corrinoid protein from Sporomusa ovata [12]. The sequence Asp-x-His-x-x-Gly containing the coordi- How are the Co–C bonds of the coenzymes activated in dif- nating histidine residue is conserved in these enzymes ferent ways? How are highly reactive radical intermediates [7] as well as in methyleneglutarate mutase [13]. The 998 Structure 1999, Vol 7 No 8 Figure 1 (a) (b) Adenosylcobalamin and the reaction NH 2 H X X mechanism catalyzed by adenosylcobalamin- N • N CC CC dependent enzymes. (a) Structure of N adenosylcobalamin (coenzyme B12). (b) The HO HO N H2NCO minimal mechanism for adenosylcobalamin- HO OH HO OH O dependent rearrangements. [Co], cobalamin; Group X migration Ade, 9-adeninyl; X, a generic migrating group. O Ade O Ade CONH CH2 CH3 2 • (c) The reaction catalyzed by diol dehydratase. [Co] [Co] N + The hydroxyl group migrates from C(2) to C(1). Co N XH X N N H2NCO • C C CC N CONH H NCO 2 2 CONH2 O N (c) OH O HN R CHCH2OH O OH RCH2CHO + H2O OH O – P O (R = CH3, H, CH2OH) O Structure reactivity of cobalamin has been proposed to be modu- interaction between the two α subunits exclusively con- lated by a proton relay through the Co-His-Asp-Ser tributes to dimerization of the heterotrimer. Two β and quartet in methionine synthase [7] or by movement of the two γ subunits are separately bound by the two α sub- Co-linked histidine in methylmalonyl-CoA mutase [8]. In units. The structure revealed explains the following bio- contrast, the above-mentioned consensus sequence is not chemical observations very well. When diol dehydratase conserved in the other four adenosylcobalamin-depen- is subjected to diethylaminoethyl (DEAE) cellulose dent enzymes including diol dehydratase [14–17] column chromatography in the absence of substrate, it (Figure 1c). EPR spectroscopic evidence for the axial dissociates into two dissimilar protein components, desig- coordination of 5,6-dimethylbenzimidazole to the cobalt nated F and S [22,23]. Components F and S have been atom in the protein-bound adenosylcobalamin has identified as the monomeric β subunit and the dimeric α γ recently been reported using adenosylcobalmin or its 2 2 complex, respectively [21]. analog with a 15N-labeled base [18,19]. The solution structure of a cobamide-binding protein (MutS) of Each heterotrimer binds one molecule of cyanocobalamin. Clostridium tetanomorphum has recently been determined The ribbon model of each subunit is shown separately in by nuclear magnetic resonance (NMR) [20]. Figure 2b. Each subunit is drawn with cyanocobalamin, K+ and propanediol in order to depict the spatial relation- Here we report the crystal structure of the diol dehy- ship between them. The cyanocobalamin molecule is dratase–cyanocobalamin complex and from it propose the located between the α and β subunits, the cyano group, an mechanism of action of diol dehydratase. This is the first upper (Coβ) ligand, being oriented to the direction of the α reported structure of a B12 enzyme that does not contain subunit. It is worth noting that the cyano group could the Asp-x-His-x-x-Gly motif. It reveals the ‘base-on’ mode not be located in the electron-density maps, and this of cobalamin binding and a characteristic molecular appa- observation will be discussed in a later section. One of the ratus for the radical reaction catalyzed by this enzyme. most striking features of this structure is that the lower Unique direct interactions of the two hydroxyl groups of (Coβ) ligand, 5,6-dimethylbenzimidazole nucleotide the substrate with K+ are another striking feature moiety, is coordinated to the cobalt atom in the corrin ring. described here. This is a remarkable new finding because in methionine synthase [7], methylmalonyl-CoA mutase [8], and gluta- Results and discussion mate mutase [11], the 5,6-dimethylbenzimidazole ligand Overall structure is displaced from the cobalt and accommodated in an Diol dehydratase exists in the dimeric form of hetero- extended form as a so-called nucleotide tail. The imida- αβγ trimer ( )2. This architecture agrees with the subunit zole group of the histidine residue in the above-men- structure deduced from molecular weight determination tioned consensus sequence of these proteins is ligated to [21]. The dimeric form has a noncrystallographic twofold the cobalt atom. The diol dehydratase–cyanocobalamin axis. Figure 2a shows a stereo pair of the mix of wire and complex therefore presents a quite new structure among γ schematic models of the dimeric form viewed along a B12 enzymes. The subunit is located far from the cofac- noncrystallographic twofold axis. It is evident that the tor and is in full contact with the α subunit. The role of Research Article Structure of B12-dependent diol dehydratase Shibata et al. 999 Figure 2 Structure of the diol dehydratase—cyanocobalamin complex. (a) Stereo drawing of the diol dehydratase–cyanocobalamin complex showing the dimeric form of the αβγ heterotrimer. Each trimer is crystallographically independent and is related by a noncrystallographic twofold axis. Colors ranging from crimson to gray are used for the α subunit, from khaki to green for the β subunit, and from cyan to violet for the γ subunit. Cyanocobalamin is located in the interface between the α and β subunits. (b) Each subunit is drawn separately in order to show the spatial relationship between each subunit and the active site. This clearly shows that the α and β subunits are in close contact with cyanocobalamin, whereas the γ subunit forms no contact with it.
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