Structural Basis for the Activation of the Cobalt-Carbon Bond and Control of the Adenosyl Radical in Coenzyme B12 Catalysis.

Adenosylcobalamin (AdoCbl), or coenzyme B12 , 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 B12 catalysis. We outline here our results obtained with these eliminating isomerases and compare them with those obtained with other radical B12 enzymes.

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12 -Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase.

The X-ray structures of coenzyme B12 (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. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.

Reactivating chaperones for coenzyme B12-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B12-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 B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.

Coenzyme B12-dependent eliminases: Diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B12-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. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.

Direct Participation of a Peripheral Side Chain of a Corrin Ring in Coenzyme†B12 Catalysis.

The crystal structures of the B12 -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. These findings guide the engineering of enzymes for improved catalysis and producing useful chemicals by utilizing the high reactivity of radical species.

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. As reported recently (T. Toraya, T. Kida, A. Kuyama, S. Matsuda, S. Tanaka, Y. Komatsu, T. Tsurukai, Biochem. Biophys. Res. Commun. 485 (2017) 41-46), it became possible to detect unknown 1-MeAde binders of starfish oocytes by immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection. We designed and synthesized water-soluble and insoluble polymer-bound 1-MeAde derivatives. A water-soluble polymer-bound 1-MeAde derivative, in which 1-MeAde is bound to dextran through an N6-substituent, triggered the germinal-vesicle breakdown toward follicle-free oocytes, dejellied oocytes, and denuded oocytes. This is consistent with the idea that putative 1-MeAde receptors are located on the cell surface of starfish oocytes. A water-insoluble polymer-bound 1-MeAde derivative, in which 1-MeAde is bound to Sepharose 4B through an N6-substituent, served as an effective affinity adsorbent for the partial purification of a 1-MeAde binder with Mr of 47.5 K that might be a possible candidate of the maturation-inducing hormone receptors of starfish oocytes.

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.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N6-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with Mr of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors.

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. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.

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. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes­i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here.

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. Spectroscopic and electrophoretic properties were consistent with the σ-donor strength or trans-effect increasing in the order: formyl

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. All five residues were important for the high-affinity binding of the substrate and for catalysis. The -COO(-) group at residue α287 was absolutely required for activity and coenzyme Co-C bond cleavage, and there was a spatially optimal position for it, suggesting that Gluα287 contributes to Co-C bond homolysis, stabilizes the transition state for the migration of NH2 from C2 to C1 through partial deprotonation of spectator OH, and functions as a base in the elimination of ammonia. A positive charge and/or the hydrogen bond at position α160 and the hydrogen bonds at positions α162 and α193 with the substrate are important for catalysis and for preventing a radical intermediate from undergoing side reactions. Argα160 would stabilize the trigonal transition state in NH2 migration by electrostatic catalysis and hydrogen bonding with spectator OH. Asnα193 would contribute to maintaining the appropriate position and direction of the guanidinium group of Argα160, as well. Hydrogen bond acceptors were necessary at position α162, but hydrogen bond donors were rather harmful. Glnα162 might stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3(+). The activity was very sensitive to the position of -COO(-) at α362. Aspα362 would assist Co-C bond homolysis indirectly and stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3(+) and electrostatic interaction.

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. The photoaffinity labeling of oocyte membrane fractions, followed by immunoblotting analysis with anti-1-MeAde antibody, results in the detection of an almost single protein band. This 1-MeAde-binding protein might be a possible candidate of the maturation-inducing hormone receptor of starfish.

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. Another important point is that adenosylcobalamin-dependent radical enzymes are prone to mechanism-based irreversible inactivation during catalysis and have their own chaperones for the maintenance of catalytic activities. This review will highlight biochemical, structural, and computational studies with special emphases on radical catalysis and reactivating chaperones of these enzymes.

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. Although Dα413N showed ATPase activity, it did not mediate cyanocobalamin (CN-Cbl) release from the DD·CN-Cbl complex in the presence of ATP or ADP and formed a tight complex with apoDD even in the presence of ATP, suggesting the involvement of Aspα413 in the nucleotide switch. In contrast, Dα8N showed very low ATPase activity and did not mediate CN-Cbl release from the complex in the presence of ATP, but it did cause about 50% release in the presence of ADP. The complex formation of this mutant with DD was partially reversed by ATP, suggesting that Aspα8 is involved in the ATPase activity but only partially in the nucleotide switch. Among DD-Rs mutated at the Mg(2+)-binding site, only Eß31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R ß subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R ß and DD ß subunits, Gluß97 of DD would coordinate to Mg(2+). The complex of Eß97Q DD with CN-Cbl was not activated by wild-type DD-R. No complex was formed between this mutant and wild-type DD-R, indicating that the coordination of Gluß97 to Mg(2+) is essential for subunit swapping and therefore for (re)activation.

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. One is predominantly responsible for the product-forming reaction (G(R) conformation), and the other primarily contributes to inactivation (G(S) conformation). Reactant radical is converted into a product and byproduct in the product-forming reaction and inactivation, respectively. The OH group migrates from C2 to C1 in the product-forming reaction, whereas the transfer of a hydrogen from the 3-OH group of glycerol to C1 takes place during the inactivation. The activation barrier of the hydrogen transfer does not depend on the substrate-binding conformation. On the other hand, the activation barrier of OH group migration is sensitive to conformation and is 4.5 kcal/mol lower in the G(R) conformation than in the G(S) conformation. In the OH group migration, Glu170 plays a critical role in stabilizing the reactant radical in the G(S) conformation. Moreover, the hydrogen bonding interaction between Ser301 and the 3-OH group of glycerol lowers the activation barrier in G(R)-TS2. As a result, the difference in energy between the hydrogen transfer and the OH group migration is reduced in the G(S) conformation, which shows that the inactivation is favored in the G(S) conformation.

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. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

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. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the G(S) conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k(inact) of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. k(cat) /k(inact) showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ. Structured digital abstract • Diol dehydrase gamma subunit, Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X-ray crystallography (View interaction).

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. The activation energy for the OH group migration, which is essential in the conversion of diols to corresponding aldehydes, is sensitive to the identity of the metal ion. For example, the spectator OH group of substrate is fully deprotonated by Glu170 in the transition state for the OH group migration in the Ca-contained QM/MM model, and therefore the barrier height is significantly decreased in the model having Ca(2+) ion. On the other hand, the deprotonation of the spectator OH group cannot effectively be triggered by the K(+) ion. Moreover, in the hydrogen recombination, the most energy-demanding step is more favorable in the Ca-contained model. The proposal that the Ca(2+) ion should be involved in the substrate-binding site is consistent with an observed large deuterium kinetic isotope effect of 10, which indicates that C-H bond activation is involved in the rate-determining step. Asp335 is found to have a strong anticatalytic effect on the OH group migration despite its important role in substrate binding. The synergistic interplay of the O-C bond cleavage by Ca(2+) ion and the deprotonation of the spectator OH group by Glu170 is required to overcome the anticatalytic effect of Asp335.