The 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.

This is the first crystallographic indication of the 'base-on' mode of cobalamin binding. An unusually long cobalt-base bond seems to favor homolytic 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.

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The structure of the adenine-ring-binding site provides a molecular basis for the strict specificity of diol dehydratase for the coenzyme adenosyl group. The superimposition of the structure of the free coenzyme on that of enzyme-bound adeninylpentylcobalamin demonstrated that the tight enzyme-coenzyme interactions at both the cobalamin moiety and adenine ring of the adenosyl group would inevitably lead to cleavage of the cobalt-carbon bond. Rotation of the ribose moiety around the glycosidic linkage makes the 5'-carbon radical accessible to the hydrogen atom of the substrate to be abstracted.

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably a 2 b 2 c 2 . When (R)-and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (K m(R) /K m(S) ¼ 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 Å resolution. The enzyme exists as a dimer of the abc heterotrimer. Cobalamin is bound at the interface between the a and b subunits in the so-called Ôbase-onÕ mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (b/a) 8 barrel that was formed by a central region of the a subunit. The substrate propane-1,2-diol and essential cofactor K + are bound inside the (b/a) 8 barrel above the corrin ring of cobalamin. K + is heptacoordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the a and b subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.Keywords: coenzyme B 12 ; adenosylcobalamin; glycerol dehydratase; crystal structure; radical enzyme catalysis.Adenosylcobalamin is one of the most unique compounds in nature. It is a water-soluble organometallic compound possessing a Co-C r bond and serves as a cofactor for enzymatic radical reactions including carbon skeleton rearrangements, heteroatom eliminations and intramolecular amino group migrations [1]. Diol dehydratase (EC 4.2.1.28) of Klebsiella oxytoca is an adenosylcobalamin (AdoCbl 1 ) dependent enzyme that catalyzes the conversions of 1,2-diols, such as propane-1,2-diol, glycerol, and 1,2-ethanediol, to the corresponding aldehydes [2,3] (Fig. 1). This enzyme has been studied intensively to establish the mechanism of action of AdoCbl [4][5][6][7]. The structurefunction relationship of the coenzyme has also been investigated extensively with this enzyme [5][6][7][8]. Recently, we have reported the three-dimensional structures of its complexes with cyanocobalamin [9] and adeninylpentylcobalamin [10] and theoretical calculations of the entire energy profile along the reaction pathway with a simplifie...

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Substrate binding triggers catalytic radical formation through the cobalt-carbon bond homolysis in coenzyme B12-dependent enzymes. We have determined the crystal structure of the substrate-free form of Klebsiella oxytoca diol dehydratase*cyanocobalamin complex at 1.85 A resolution. The structure contains two units of the heterotrimer consisting of alpha, beta, and gamma subunits. As compared with the structure of its substrate-bound form, the beta subunits are tilted by approximately 3 degrees and cobalamin is also tilted so that pyrrole rings A and D are significantly lifted up toward the substrate-binding site, whereas pyrrole rings B and C are only slightly lifted up. The structure revealed that the potassium ion in the substrate-binding site of the substrate-free enzyme is also heptacoordinated; that is, two oxygen atoms of two water molecules coordinate to it instead of the substrate hydroxyls. A modeling study in which the structures of both the cobalamin moiety and the adenine ring of the coenzyme were superimposed onto those of the enzyme-bound cyanocobalamin and the adenine ring-binding pocket, respectively, demonstrated that the distortions of the Co-C bond in the substrate-free form are already marked but slightly smaller than those in the substrate-bound form. It was thus strongly suggested that the Co-C bond becomes largely activated (labilized) when the coenzyme binds to the apoenzyme even in the absence of substrate and undergoes homolysis through the substrate-induced conformational changes of the enzyme. Kinetic coupling of Co-C bond homolysis with hydrogen abstraction from the substrate shifts the equilibrium to dissociation.

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