The crystal structure of coenzyme B₁₂‐dependent glycerol dehydratase in complex with cobalamin and propane‐1,2‐diol

Abstract

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...

Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase

“…There are four helpful examples: glutamate mutase (PDB ID code 1I9C) from Clostridium cochlearium (10), methylmalonyCoA mutase (MMCoA mutase) from P. shermanii (9) (PDB ID code 1REQ), diol dehydratase (40) (PDB ID codes 1DIO and 1EEX), and glycerol dehydratase (PDB ID code 1IWP) from Klebsiella pneumoniae (41). Interactions of cobalamin with these proteins all follow a pattern in which the methyl groups of rings C and D, at the hydrophobic rim of the corrin macrocycle (Fig.…”

Construction and Characterization of Hybrid Dehydratases between Adenosylcobalamin-Dependent Diol and Glycerol Dehydratases

“…Inability to form these hybrid complexes might be partly due to the loss of mutual interaction between heterologous ␣D and ␥G subunits, because the homologous ␣ and ␥ subunits form a tight complex (components S and A in diol dehydratase (6) and glycerol dehydratase (7), respectively). Tight interactions between ␣ and ␥ subunits were revealed by the X-ray structures of diol and glycerol dehydratases (7,24). Co-existence and mutual interaction of ␣ and ␥ subunits are required for correct folding of diol dehydratase (6).…”

“…These regions of ␤ and ␥ subunits of diol dehydratase lower the solubility of diol dehydratase ( 23 ). The X-ray structures ( 7,24,25 ) revealed that both enzymes exist as ( ␣␤␥ ) 2 complexes whose overall structures are strikingly similar. The structures of the active sites of both enzymes are also similar-that is, the K ϩ ion is bound in the inner part of the so-called TIM (triose phosphate isomerase) barrel of the ␣ subunit, and the two hydroxyl groups of substrate 1,2-propanediol are directly coordinated to this K ϩ ion.…”

“…They consist of the three different subunits, ␣ ( Mr 60,000-61,000), ␤ ( Mr 21,000-24,000), and ␥ ( Mr 16,000-19,000), and dissociate into two dissimilar protein components in the absence of substrate ( 4,5 ). Large and small components correspond to the ␣ 2 ␥ 2 complex and the ␤ subunit, respectively ( 6,7 ). Their catalytic properties are similar, but they are different in the binding affinity for AdoCbl ( 8,9 ), substrate specificity ( 1-3, 10, 11 ), susceptibility to suicide inactivation by glycerol ( 2,9,10 ), monovalent cation specificity ( 1,11,12 ), and immunochemical reactivity toward anti-diol dehydratase antiserum ( 11 ) (for review, see Refs.…”

“…The ␥ subunit is attached on the ␣ subunit apart from the substrate-and coenzyme-binding sites. The amino acid residues that interact directly with K ϩ and/or 1,2-propanediol are conserved between these two enzymes ( 7,24 ), although the substrate specificity and the monovalent cation specificity are somewhat different between them. To help the understanding of the role of each subunit in the catalysis, a study of the hybrid enzymes between diol and glycerol dehydratases may be useful, but not yet reported so far.…”