Energetic feasibility of hydrogen abstraction and recombination in coenzyme B(12)-dependent diol dehydratase reaction.

Coenzyme B(12) serves as a cofactor for enzymatic radical reactions. The essential steps in all the coenzyme B(12)-dependent rearrangements are two hydrogen abstraction steps: hydrogen abstraction of the adenosyl radical from substrates, and hydrogen back-abstraction (recombination) of a product-derived radical from 5'-deoxyadenosine. The energetic feasibility of these hydrogen abstraction steps in the diol dehyratase reaction was examined by theoretical calculations with a protein-free, simplified model at the B3LYP/6-311G* level of density functional theory. Activation energies for the hydrogen abstraction and recombination with 1,2-propanediol as substrate are 9.0 and 15.1 kcal/mol, respectively, and essentially not affected by coordination of the substrate and the radical intermediate to K+. Since these energies can be considered to be supplied by the substrate-binding energy, the computational results with this simplified model indicate that the hydrogen abstraction and recombination in the coenzyme B(12)-dependent diol dehydratase reaction are energetically feasible.

Radical production simulated by photoirradiation of the diol dehydratase-adeninylpentylcobalamin complex.

In the course of structural studies of diol dehydratase-cobalamin complexes, it was found that the electron density corresponding to the cyano group of the enzyme-bound cyanocobalamin is almost not observable at room temperature and very low even at cryogenic temperatures, suggesting its dissociation from the Co atom upon X-ray irradiation. On the contrary, the adenine moiety of the enzyme-bound adeninylpentylcobalamin was clearly located in the electron density map. When the enzyme-adeninylpentylcobalamin complex was illuminated with visible light, the electron density between the C5' and Co atoms disappeared, and the temperature factors of the atoms comprising the pentamethylene group became much larger than those in the dark. This indicates a Co-C bond cleavage and that the adenine moiety remains held by hydrogen bonds with some residues in the enzyme. Thus, the formation of an adenine-anchored radical upon illumination was demonstrated crystallographically with this complex. These observations clearly indicate that homolysis of the Co-C bond of alkylcobalamin takes place upon illumination with visible light but is not readily cleaved during X-ray irradiation.

Revised sequence and expression of cyclin B cDNA from the starfish Asterina pectinifera.

Cyclin B cDNA was cloned from the ovary of the starfish Asterina pectinifera and analyzed by RT-PCR and 3'- and 5'-RACE techniques. The cDNA consists of a 0.13-kb upstream untranslated region, a 1.22-kb coding region, and a 0.86-kb downstream untranslated region. The open reading frame encoded a polypeptide of 404 amino acid residues with a calculated molecular weight of 45,692. All the characteristic sequences, such as destruction and cyclin boxes, cyclin B motif, and cytoplasmic retention and nuclear export signals, were found in the newly cloned cyclin B cDNA. The deduced amino acid sequence of the cyclin B cDNA was highly homologous in the middle and carboxy terminal regions to that from mature eggs of the same organism, but quite different in the amino terminal region. Evidence was obtained which suggested that this cyclin B is expressed in immature and maturing oocytes and is the same as that cloned from mature eggs.

Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase.

Adenosylcobalamin-dependent glycerol dehydratase undergoes mechanism-based inactivation by its physiological substrate glycerol. We identified two genes (gdrAB) of Klebsiella pneumoniae for a glycerol dehydratase-reactivating factor (Tobimatsu, T., Kajiura, H., Yunoki, M., Azuma, M., and Toraya, T. (1999) J. Bacteriol. 181, 4110-4113). Recombinant GdrA and GdrB proteins formed a tight complex of (GdrA)(2)(GdrB)(2), which is a putative reactivating factor. The purified factor reactivated the glycerol-inactivated and O(2)-inactivated glycerol dehydratases as well as activated the enzyme-cyanocobalamin complex in vitro in the presence of ATP, Mg(2+), and adenosylcobalamin. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins in the presence of ATP and Mg(2+) through intermediate formation of apoenzyme. The factor showed extremely low ATP-hydrolyzing activity and formed a tight complex with apoenzyme in the presence of ADP. Incubation of the enzyme-cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin. The resulting tight inactive complex of apoenzyme with the factor dissociated upon incubation with ATP, forming functional apoenzyme and a low affinity form of factor. Thus, it was established that the reactivation of the inactivated holoenzymes takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme-factor complex. We propose that the glycerol dehydratase-reactivating factor is a molecular chaperone that participates in reactivation of the inactivated enzymes.

Orally tolerant CD4 T cells respond poorly to antigenic stimulation but strongly to direct stimulation of intracellular signaling pathways.

The response of splenic CD4 T cells from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic mice after long-term feeding of a diet containing this antigen was examined. These CD4 T cells exhibited a decreased response to OVA peptide stimulation, in terms of proliferation, interleukin-2 secretion, and CD40 ligand expression, compared to those from mice fed a control diet lacking OVA, demonstrating that oral tolerance of T cells had been induced through oral intake of the antigen. We investigated the intracellular signaling pathways, which were Ca/CN cascade and Ras/MAPK cascade, of these tolerant CD4 T cells using phorbol-12-myristate-13-acetate (PMA) and ionomycin, which are known to directly stimulate these pathways. In contrast to the decreased response to TCR stimulation by OVA peptide, it was shown that the response of splenic CD4 T cells to these reagents in the state of oral tolerance was stronger. These results suggest that splenic CD4 T cells in the state of oral tolerance have an impairment in signaling, in which signals are not transmitted from the TCR to downstream signaling pathways, and have impairments in the vicinity of TCR.

Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase.

Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase.

Radical catalysis of B12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases.

Enzymatic radical catalysis is defined as a mechanism of catalysis by which enzymes catalyze chemically difficult reactions by utilizing the high reactivity of free radicals. Adenosylcobalamin (coenzyme B12) serves as a cofactor for enzymatic radical reactions. The recent structural analysis of adenosylcobalamin-dependent diol dehydratase revealed that the substrate 1,2-propanediol and an essential potassium ion are located inside a (beta/alpha)8 barrel. Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity. Cobalamin bound in the base-on mode covers the cavity to isolate the active site from solvent. Based on the three-dimensional structure and theoretical calculations, a new mechanism for diol dehydratase is proposed in which the potassium ion plays a direct role in the catalysis. The mechanisms for generation of a catalytic radical by homolysis of the coenzyme Co-C bond and for protection of radical intermediates from undesired side reactions during catalysis are discussed based on the structure. The reactivating factors for diol and glycerol dehydratases have been identified. These factors are a new type of molecular chaperone which participate in reactivation of the inactivated holoenzymes by mediating ATP-dependent exchange of the modified coenzyme for free intact coenzyme.

Extremely low activity of methionine synthase in vitamin B-12-deficient rats may be related to effects on coenzyme stabilization rather than to changes in coenzyme induction.

Severely vitamin B-12 (B-12)-deficient rats were produced by feeding a B-12-deficient diet. The status of B-12 deficiency was confirmed by an increase in urinary methylmalonate excretion and decreases in liver B-12 concentrations and cobalamin-dependent methionine synthase activity. Rat liver methionine synthase existed almost exclusively as the holoenzyme. In B-12-deficient rats, the level of methionine synthase protein was lower, although the mRNA level was not significantly different from that of control rats. When methylcobalamin, the coenzyme for methionine synthase, was administered to the B-12-deficient rats, growth, liver B-12 concentrations and urinary excretion of methylmalonate were reversed although not always to control (B-12-sufficient) levels in a short period. During this recovery process, methionine synthase activity and its protein level increased, whereas the mRNA level was unaffected. We reported previously that rat apomethionine synthase is very unstable and is stabilized by forming a complex with methylcobalamin. Thus, the extremely low activity of methionine synthase in B-12-deficient rats may be related to effects on "coenzyme stabilization" (stabilization of the enzyme by cobalamin binding) rather than to changes in "coenzyme induction."

How a protein generates a catalytic radical from coenzyme B(12): X-ray structure of a diol-dehydratase-adeninylpentylcobalamin complex.

BACKGROUND: Adenosylcobalamin (coenzyme B(12)) serves as a cofactor for enzymatic radical reactions. The adenosyl radical, a catalytic radical in these reactions, is formed by homolysis of the cobalt-carbon bond of the coenzyme, although the mechanism of cleavage of its organometallic bond remains unsolved. RESULTS: We determined the three-dimensional structures of diol dehydratase complexed with adeninylpentylcobalamin and with cyanocobalamin at 1.7 A and 1.9 A resolution, respectively, at cryogenic temperatures. In the adeninylpentylcobalamin complex, the adenine ring is bound parallel to the corrin ring as in the free form and methylmalonyl-CoA-mutase-bound coenzyme, but with the other side facing pyrrole ring C. All of its nitrogen atoms except for N(9) are hydrogen-bonded to mainchain amide oxygen and amide nitrogen atoms, a sidechain hydroxyl group, and a water molecule. As compared with the cyanocobalamin complex, the sidechain of Seralpha224 rotates by 120 degrees to hydrogen bond with N(3) of the adenine ring. CONCLUSIONS: 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.

Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase.

Rat methionine synthase was expressed chiefly as apoenzyme in recombinant baculovirus-infected insect cells (Yamada, K., Tobimatsu, T., and Toraya, T. (1998) Biosci. Biotech. Biochem. 62, 2155-2160). The apoenzyme produced was very unstable, and therefore, after complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K(m) values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob(II)alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium(III) and S-adenosylmethionine. The rate of oxidative inactivation of the enzyme in the absence of S-adenosylmethionine was slower with a stronger reducing agent like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective or were effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by photoirradiation.

Mechanism of reactivation of coenzyme B12-dependent diol dehydratase by a molecular chaperone-like reactivating factor.

The mechanism of reactivation of diol dehydratase by its reactivating factor was investigated in vitro by using enzyme. cyanocobalamin complex as a model for inactivated holoenzyme. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins through intermediate formation of apoenzyme. The factor showed extremely low but distinct ATP-hydrolyzing activity. It formed a tight complex with apoenzyme in the presence of ADP but not at all in the presence of ATP. Incubation of the enzyme.cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin, leaving the tight apoenzyme-reactivating factor complex. Although the resulting complex was inactive even in the presence of added adenosylcobalamin, it dissociated by incubation with ATP, forming the apoenzyme, which was reconstitutable into active holoenzyme with added coenzyme. Thus, it was established that the reactivation of the inactivated holoenzyme by the factor in the presence of ATP and Mg2+ takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme.factor complex. ATP plays dual roles as a precursor of ADP in the first step and as an effector to change the factor into the low-affinity form for diol dehydratase. The enzyme-bound adenosylcobalamin was also susceptible to exchange with free adeninylpentylcobalamin, although to a much lesser degree. The mechanism for discrimination of adenine-containing cobalamins from adenine-lacking cobalamins was explained in terms of formation equilibrium constants of the cobalamin.enzyme.reactivating factor ternary complexes. We propose that the reactivating factor is a new type of molecular chaperone that participates in reactivation of the inactivated enzymes.

Direct participation of potassium ion in the catalysis of coenzyme B(12)-dependent diol dehydratase.

The direct ion-dipolar interactions between potassium ion (K(+)) and the two hydroxyl groups of the substrate are the most striking feature of the crystal structure of coenzyme B(12)-dependent diol dehydratase. We carried out density-functional-theory computations to determine whether K(+) can assist the 1,2-shift of the hydroxyl group in the substrate-derived radical. Between a stepwise abstraction/recombination reaction proceeding via a direct hydroxide abstraction by K(+) and a concerted hydroxyl group migration assisted by K(+), only a transition state for the latter concerted mechanism was found from our computations. The barrier height for the transition state from the complexed radical decreases by only 2.3 kcal/mol upon coordination of the migrating hydroxyl group to K(+), which corresponds to a 42-fold rate acceleration at 37 degrees C. The net binding energy upon replacement of the K(+)-bound water for substrate was calculated to be 10.7 kcal/mol. It can be considered that such a large binding energy is at least partly used for the substrate-induced conformational changes in the enzyme that trigger the homolytic cleavage of the Co-C bond of the coenzyme and the subsequent catalysis by a radical mechanism. We propose here a new mechanism for diol dehydratase in which K(+) plays a direct role in the catalysis.

A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase.

BACKGROUND: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B12) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt-carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an alpha2beta2gamma2 heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase-cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme. RESULTS: The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 A resolution. The enzyme exists as a dimer of heterotrimers (alphabetagamma)2. The cobalamin molecule is bound between the alpha and beta subunits in the 'base-on' mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The alpha subunit includes a (beta/alpha)8 barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion. CONCLUSIONS: 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.

Identification and expression of the genes encoding a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase.

Adenosylcobalamin-dependent glycerol dehydratase undergoes inactivation by glycerol, the physiological substrate, during catalysis. In permeabilized cells of Klebsiella pneumoniae, the inactivated enzyme is reactivated in the presence of ATP, Mg2+, and adenosylcobalamin. We identified the two open reading frames as the genes for a reactivating factor for glycerol dehydratase and designated them gdrA and gdrB. The reactivation of the inactivated glycerol dehydratase by the gene products was confirmed in permeabilized recombinant Escherichia coli cells coexpressing GdrA and GdrB proteins with glycerol dehydratase.

Crystallization and preliminary x-ray study of two crystal forms of Klebsiella oxytoca diol dehydratase-cyanocobalamin complex.

Two crystal forms of Klebsiella oxytoca diol dehydratase complexed with cyanocobalamin have been obtained and preliminary crystallographic experiments have been performed. The crystals belong to two different space groups, depending on the crystallization conditions. One crystal (form I) belongs to space group P212121 with unit-cell parameters a = 76.2, b = 122.3, c = 209. 6 A, and diffracts to 2.2 A resolution using an X-ray beam from a synchrotron radiation source. The other crystal (form II) belongs to space group P21 with unit-cell parameters a = 75.4, b = 132.7, c = 298.8 A, beta = 91.9 degrees, and diffracts to 3.0 A resolution. For the purpose of structure determination, a heavy-atom derivative search was carried out and some mercuric derivatives were found to be promising. Structure analysis by the multiple isomorphous replacement method is now under way.

A reactivating factor for coenzyme B12-dependent diol dehydratase.

Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicide inactivation by glycerol, a physiological substrate. The coenzyme is modified through irreversible cleavage of its cobalt-carbon bond, resulting in inactivation of the enzyme by tight binding of the modified coenzyme to the active site. Recombinant DdrA and DdrB proteins of K. oxytoca were co-purified to homogeneity from cell-free extracts of Escherichia coli overexpressing the ddrAB genes. They existed as a tight complex, i.e. a putative reactivating factor, with an apparent molecular weight of 150,000. The factor consists of equimolar amounts of the two subunits with Mr of 64,000 (A) and 14,000 (B), encoded by the ddrA and ddrB genes, respectively. Therefore, its subunit structure is most likely A2B2. The factor not only reactivated glycerol-inactivated and O2-inactivated holoenzymes but also activated enzyme-cyanocobalamin complex in the presence of free adenosylcobalamin, ATP, and Mg2+. The reactivating factor mediated ATP-dependent exchange of the enzyme-bound cyanocobalamin for free 5-adeninylpentylcobalamin in the presence of ATP and Mg2+, but the reverse was not the case. Thus, it can be concluded that the inactivated holoenzyme becomes reactivated by exchange of the enzyme-bound, adenine-lacking cobalamins for free adenosylcobalamin, an adenine-containing cobalamin.

Molecular cloning, sequencing and characterization of the genes for adenosylcobalamin-dependent diol dehydratase of Klebsiella pneumoniae.

Klebsiella pneumoniae and some of the other Enterobacteriaceae form both diol dehydratase and glycerol dehydratase in response to growth substrates. To compare these enzymes produced by the same bacterium, the pdd genes of K. pneumoniae encoding adenosylcobalamin-dependent diol dehydratase were cloned and sequenced. The sequential three open reading frames (pddA, pddB, and pddC genes) encoded polypeptides of 554, 228, and 174 amino acid residues with predicted molecular weights of 60,379(alpha), 24,401(beta), and 19,489(gamma), respectively. The deduced amino acid sequences of the subunits were 84-100% and 54-71% identical with those reported for diol dehydratases and glycerol dehydratases, respectively.

EPR spectroscopic evidence for the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydratase by coenzyme analogs.

EPR spectra were measured upon incubation of the complex of diol dehydratase with coenzyme analogs in the presence of 1,2-propanediol, a physiological substrate. When the analog in which the D-ribose moiety of the nucleotide loop was replaced by a trimethylene group was used as coenzyme, essentially the same EPR spectrum as that with adenosylcobalamin was obtained. The higher-field doublet and the lower-field broad signals derived from an organic radical and low-spin Co(II) of cob(II)alamin, respectively, were observed. With the imidazolyl counterpart, base-on cob(II)alamin-like species accumulated, but signals due to an organic radical quickly disappeared. When a coenzyme analog lacking the nucleotide moiety was incubated with apoenzyme in the presence of substrate, the EPR spectrum resembling cob(II)inamide was obtained, but no signals due to an organic radical were observed. From these results, it was concluded that the extinction of organic radical intermediates results in inactivation of the enzyme by these coenzyme analogs. Upon suicide inactivation with a [15N2]imidazolyl analog, the octet signals due to Co(II) showed superhyperfine splitting into doublets, indicating axial coordination of 5,6-dimethylbenzimidazole to the cobalamin bound to diol dehydratase.

Evidence for axial coordination of 5,6-dimethylbenzimidazole to the cobalt atom of adenosylcobalamin bound to diol dehydratase.

It was demonstrated by electron paramagnetic resonance (EPR) spectroscopy that organic radical intermediates disappeared and cob(II)alamin accumulated upon suicide inactivation of diol dehydratase by 2-methyl-1,2-propanediol. The resulting EPR spectra showed that the eight hyperfine lines due to the divalent cobalt atom of cob(II)alamin further split into triplets by the superhyperfine coupling to the 14N nucleus. Essentially the same superhyperfine splitting of the octet into triplets was observed with [14N]- and [15N]apoenzyme. When the adenosyl form of [14N2]- and [15N2]imidazolyl analogues of the coenzyme [Toraya, T., and Ishida, A. (1991) J. Biol. Chem. 266, 5430-5437] was used with unlabeled apoenzyme, the octet showed superhyperfine splitting into triplets and doublets, respectively. Therefore, it was concluded that cobalamin is bound to this enzyme with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. This conclusion is consistent with the fact that the consensus sequence forming part of a cobalamin-binding motif, conserved in methionine synthase and some of the other cobalamin enzymes, was not found in the deduced amino acid sequences of the subunits of diol dehydratase. Adenosylcobinamide methyl phosphate, a coenzyme analogue lacking the nucleotide moiety, underwent cleavage of the cobalt-carbon bond upon binding to the enzyme in the presence of substrate, forming a cob(II)inamide derivative without nitrogenous base coordination, as judged by EPR and optical spectroscopy. Therefore, this analogue may be a useful probe for determining whether the replacement of the 5, 6-dimethylbenzimidazole ligand by a histidine residue takes place upon binding of cobalamin to proteins.