The action of coenzyme B12-dependent diol dehydratase on 3,3,3-trifluoro-1,2-propanediol results in elimination of all the fluorides with formation of acetaldehyde.

3,3,3-Trifluoro-1,2-propanediol undergoes complete defluorination in two distinct steps: first, the conversion into 3,3,3-trifluoropropionaldehyde catalyzed by adenosylcobalamin (coenzyme B12)-dependent diol dehydratase; second, non-enzymatic elimination of all three fluorides from this aldehyde to afford malonic semialdehyde (3-oxopropanoic acid), which is decarboxylated to acetaldehyde. Diol dehydratase accepts 3,3,3-trifluoro-1,2-propanediol as a relatively poor substrate, albeit without significant mechanism-based inactivation of the enzyme during catalysis. Optical and electron paramagnetic resonance (EPR) spectra revealed the steady-state formation of cob(II)alamin and a substrate-derived intermediate organic radical (3,3,3-trifluoro-1,2-dihydroxyprop-1-yl).

Structural Basis for the Activation of the Cobalt-Carbon Bond and Control of the Adenosyl Radical in Coenzyme B Catalysis.

Adenosylcobalamin (AdoCbl), or coenzyme B , is a naturally occurring organometallic compound that serves as a cofactor for enzymes that catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. AdoCbl-dependent enzymes are radical enzymes that generate an adenosyl radical by homolysis of the coenzyme's cobalt-carbon (Co-C) bond for catalysis. How do the enzymes activate and cleave the Co-C bond to form the adenosyl radical? How do the enzymes utilize the high reactivity of the adenosyl radical for catalysis by suppressing undesirable side reactions? Our recent structural studies, which aimed to solve these problems with diol dehydratase and ethanolamine ammonia-lyase, established the crucial importance of the steric strain of the Co-C bond and conformational stabilization of the adenosyl radical for coenzyme B catalysis.

Structural Insights into the Very Low Activity of the Homocoenzyme B Adenosylmethylcobalamin in Coenzyme B -Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase.

The X-ray structures of coenzyme B (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states.

Reactivating chaperones for coenzyme B-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B eliminases.

Coenzyme B-dependent eliminases: Diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase.

Direct Participation of a Peripheral Side Chain of a Corrin Ring in Coenzyme B Catalysis.

The crystal structures of the B -dependent isomerases (eliminating) diol dehydratase and ethanolamine ammonia-lyase complexed with adenosylcobalamin were solved with and without substrates. The structures revealed that the peripheral a-acetamide side chain of the corrin ring directly interacts with the adenosyl group to maintain the group in the catalytic position, and that this side chain swings between the original and catalytic positions in a synchronized manner with the radical shuttling between the coenzyme and substrate/product. Mutations involving key residues that cooperatively participate in the positioning of the adenosyl group, directly or indirectly through the interaction with the a-side chain, decreased the turnover rate and increased the relative rate of irreversible inactivation caused by undesirable side reactions.

Affinity chromatography of a binder of 1-methyladenine, the maturation-inducing hormone for starfish oocytes.

Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary. They resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the maturation-inducing hormone for starfish oocytes. Putative 1-MeAde receptors have been suggested to be present on the oocyte surface, but not yet been characterized biochemically.

Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish.

Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.

Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase.

Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2.

Molecular architectures and functions of radical enzymes and their (re)activating proteins.

Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases.

Synthesis and properties of formylcobalamin and propionylcobalamin, novel acylcobalamins.

Formylcobalamin (formyl-Cbl), a C1-unit carrying corrinoid, and propionylcobalamin (propionyl-Cbl) were synthesized for the first time, and their properties were compared with those of acetylcobalamin (acetyl-Cbl). Formyl-Cbl, acetyl-Cbl, and propionyl-Cbl were decomposed by a NH2OH treatment, forming formo-, aceto-, and propionohydroxamic acids, respectively, which offers a proof for the presence of "activated" acyl groups and for their structures of Co-acyl-Cbls. These results, together with chromatographic, electrophoretic, and spectroscopic properties, indicate that the acyl-Cbls synthesized are actually formyl-Cbl, acetyl-Cbl, and propionyl-Cbl.

Catalytic roles of substrate-binding residues in coenzyme B12-dependent ethanolamine ammonia-lyase.

Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. 1-OH of the substrate is hydrogen-bonded with Gluα287, Argα160, and Asnα193 and 2-NH2 with Gluα287, Glnα162, and Aspα362. The active site somewhat resembles that of diol dehydratase.

Immunophotoaffinity labeling of the binding proteins for 1-methyladenine, an oocyte maturation-inducing hormone of starfish.

Starfish oocytes are naturally arrested at the prophase stage of the first meiotic division and resume meiosis in response to 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative receptors for 1-MeAde have not yet been characterized biochemically, although the specific binding of 1-MeAde to the isolated cortices of starfish oocytes was reported so far. Based on the structure-activity relationship of 1-MeAde analogs, we have designed a photoaffinity labeling reagent.

Cobalamin-dependent dehydratases and a deaminase: radical catalysis and reactivating chaperones.

Adenosylcobalamin, a coenzyme form of vitamin B12, is an organometallic compound that participates in about ten enzymatic reactions. These enzymes catalyze chemically challenging reactions by using a highly reactive primary carbon radical that is derived from homolysis of the coenzyme Co-C bond. Among them, diol dehydratases and ethanolamine ammonia-lyase have been most extensively studied to establish the general mechanism of adenosylcobalamin-assisted enzymatic catalysis and radical-catalyzed reactions.

Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase.

Diol dehydratase-reactivase (DD-R) is a molecular chaperone that reactivates inactivated holodiol dehydratase (DD) by cofactor exchange. Its ADP-bound and ATP-bound forms are high-affinity and low-affinity forms for DD, respectively. Among DD-Rs mutated at the nucleotide-binding site, neither the Dα8N nor Dα413N mutant was effective as a reactivase.

Inactivation mechanism of glycerol dehydration by diol dehydratase from combined quantum mechanical/molecular mechanical calculations.

Inactivation of diol dehydratase during the glycerol dehydration reaction is studied on the basis of quantum mechanical/molecular mechanical calculations. Glycerol is not a chiral compound but contains a prochiral carbon atom. Once it is bound to the active site, the enzyme adopts two binding conformations.

Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant.

Redesign of coenzyme B(12) dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols.

Coenzyme B(12) dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [Biochemistry16, 1082-1092 (1977)] reported that glycerol bound in the G(S) conformation, in which the pro-S-CH(2) OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction.

Relevance of nuclear receptor expression in a Tchreg cell line, HOZOT: RXRα and PPARγ negatively regulate IFN-γ production.

Nuclear receptors (NRs) have recently received much attention for their newly discovered roles in T cell development, as exemplified by RARα (Treg cells) and RORγt (Th17 cells). In previous studies, we characterized a new type of T cell subset, designated as Tchreg (cytotoxic, helper, and regulatory T) cells, in terms of its cytokine signature. In this study, we investigated the expression and functional relevance of NRs in Tchreg cells by performing mRNA profiling of HOZOT, a cord blood-derived Tchreg cell line.

Marked induction of CD4+CD8+ T cells with multifunctional properties by coculturing CD2+ cells with xenogeneic stromal cells.

A number of T cell subsets have been identified, and the in vitro characterization of these subsets largely depends on an appropriate induction system for each one. In previous studies, we characterized a unique T cell line, HOZOT, which possessed a CD4+CD8+ double positive (DP) phenotype and multifunctional properties including cytotoxic, helper, and regulatory functions. Therefore, this T cell subset has been termed Tchreg cells.

Catalytic roles of the metal ion in the substrate-binding site of coenzyme B12-dependent diol dehydratase.

Functions of the metal ion in the substrate-binding site of diol dehydratase are studied on the basis of quantum mechanical/molecular mechanical (QM/MM) calculations. The metal ion directly coordinates to substrate and is essential for structural retention and substrate binding. The metal ion has been originally assigned to the K(+) ion; however, QM/MM computations indicate that Ca(2+) ion is more reasonable as the metal ion because calculated Ca-O distances better fit to the coordination distances in X-ray crystal structures rather than calculated K-O distances.

How coenzyme B12-dependent ethanolamine ammonia-lyase deals with both enantiomers of 2-amino-1-propanol as substrates: structure-based rationalization.

Coenzyme B(12)-dependent ethanolamine ammonia-lyase acts on both enantiomers of the substrate 2-amino-1-propanol [Diziol, P., et al. (1980) Eur.

[Microbe-inspired system enzymology of vitamin B₁₂ metabolism].

Vitamin B₁₂ is produced only by prokaryotes and utilized by animals as an essential micronutrient. Genetic complementation analysis of cell lines from patients indicated that at least eight gene products are involved in intracellular B₁₂ metabolism and utilization. We have investigated bacterial adenosylcobalamin-dependent enzymes and elucidated their structure-based fine mechanisms.