Exploring the Biosynthetic Potential of TsrM,a B12-dependent Radical SAM Methyltransferase Catalyzing Non-radical Reactions

B₁₂-dependent radical SAM enzymes are an emerging enzyme family with approximately 200,000 proteins. These enzymes have been shown to catalyze chemically challenging reactions such as methyl transfer to sp2- and sp3-hybridized carbon atoms. However, to date we have little information regarding their complex mechanisms and their biosynthetic potential. Here we show, using X-ray absorption spectroscopy, mutagenesis and synthetic probes that the vitamin B₁₂-dependent radical SAM enzyme TsrM catalyzes not only C- but also N-methyl transfer reactions further expanding its synthetic versatility. We also demonstrate that TsrM has the unique ability to directly transfer a methyl group to the benzyl core of tryptophan, including the least reactive position C4. Collectively, our study supports that TsrM catalyzes non-radical reactions and establishes the usefulness of radical SAM enzymes for novel biosynthetic schemes including serial alkylation reactions at particularly inert C−H bonds.     

Mild olefin formation via bio-inspired vitamin B12 photocatalysis

Dehydrohalogenation, or elimination of hydrogen-halide equivalents, remains one of the simplest methods for the installation of the biologically-important olefin functionality. However, this transformation often requires harsh, strongly-basic conditions, rare noble metals, or both, limiting its applicability in the synthesis of complex molecules. Nature has pursued a complementary approach in the novel vitamin B₁₂-dependent photoreceptor CarH, where photolysis of a cobalt–carbon bond leads to selective olefin formation under mild, physiologically-relevant conditions. Herein we report a light-driven B₁₂-based catalytic system that leverages this reactivity to convert alkyl electrophiles to olefins under incredibly mild conditions using only earth abundant elements. Further, this process exhibits a high level of regioselectivity, producing terminal olefins in moderate to excellent yield and exceptional selectivity. Finally, we are able to access a hitherto-unknown transformation, remote elimination, using two cobalt catalysts in tandem to produce subterminal olefins with excellent regioselectivity. Together, we show vitamin B₁₂ to be a powerful platform for developing mild olefin-forming reactions.

Terminal or subterminal olefins can be selectively formed from alkyl electrophiles via bio-inspired vitamin B₁₂ photocatalysis.     

Dioldehydratase Binds Coenzyme B12 in the “Base-On” Mode: ESR Investigations on Cob(II)alamin

Even in the enzyme-bound state the dimethylbenzimidazole ligand in the dioldehydratase from Salmonella typhimurium remains bound to the cobalt ion in contrast to some coenzyme B₁₂-dependent enzymes. Direct, ESR spectroscopic proof for this “base-on” binding mode was obtained by using a coenzyme in which one of the nitrogen atoms of the dimethylbenzimidazole ligand was ¹⁵N labeled (see schematic representation on the right).     

Electrochemical Synthesis and Structure Analysis of Neocoenzyme B12 – An Epimer of Coenzyme B12 with a Remarkably Flexible Organometallic Group

In neocoenzyme B₁₂ (=(5′-deoxy-5′-adenosyl)-13-epicob(III)alamin; 5 ), an epimer of coenzyme B₁₂ ( 1 ), the organometallic group and a propanamide side chain of the vitamin-B₁₂ ligand compete for the same region in space. Interesting consequences for structure and organometallic reactivity of this isomer of 1 are to be expected. Neocoenzyme B₁₂ ( 5 ; 89% yield) and methyl-13-epicobalamin ( 6 ; 88% yield) were prepared from neovitamin B₁₂ ( 4 ) by electrochemical means (Fig. 3). The solution structure of the organometallic neovitamin-B₁₂ derivative 5 was analyzed by homonuclear and heteronuclear NMR spectroscopy. Comparison of the structures of 1 and 5 informed on the structural consequences of the epimerization at C(13) and revealed a remarkable flexibility of the organometallic group in 5 . In 5 , both sterically interacting functionalities (organometallic group and propanamide side chain at C(13)) adapt their conformations dynamically to avoid significant mutual clashes. As one consequence of this structural adaptation, the major conformations of 5 feature counterclockwise and clockwise reorientations of the organometallic ligand with respect to its crystallographically determined position in coenzyme B₁₂ ( 1 ). One of the dominant conformers of 5 exhibits an orientation of the organometallic functionality similar to that found in the crystal structure of the coenzyme-B₁₂-dependent methylmalonyl CoA mutase. The present NMR study also revealed the significant population of syn-conformers of the organometallic adenosine group, another remarkable feature of the solution structure of 5 .     

Antivitamins B12—A Structure‐ and Reactivity‐Based Concept

B₁₂‐antimetabolites are compounds that counteract the physiological effects of vitamin B₁₂ and related natural cobalamins. Presented here is a structure‐ and reactivity‐based concept of the specific ′antivitamins B₁₂′: it refers to analogues of vitamin B₁₂ that display high structural similarity to the vitamin and are ′locked chemically′ to prevent their metabolic conversion into the crucial organometallic B₁₂‐cofactors. Application of antivitamins B₁₂ to healthy laboratory animals is, thus, expected to induce symptoms of B₁₂‐deficiency. Antivitamins B₁₂ may, hence, be helpful in elucidating still largely puzzling pathophysiological phenomena associated with B₁₂‐deficiency, and also in recognizing physiological roles of B₁₂ that probably still remain to be discovered.     

Zur Kenntnis der β-Lysin-Mutase-Reaktion: Mechanismus und sterischer Verlauf

Investigations on the β-lysine mutase reaction: Mechanism and steric course The steric course and some mechanistic aspects of the coenzyme-B₁₂-dependent β-lysine-mutase reaction, in which (3 S)-β-lysine is converted to (3 S, 5 S)-3, 5-diaminohexanoate, have been investigated by means of tritium labelling. The reaction involves migration of an hydrogen atom from C(5) of the substrate to C(5′) of coenzyme B₁₂ and back-transfer to C(6) of the product. In the presence of [5′-³H]-coenzyme B₁₂ the enzyme catalyzes the exchange of label between the cofactor and one of the diastereotopic H-atoms at C(5) of the substrate. The exchangeable hydrogen atom is identical with the one specifically involved in the migration reaction. Degradation of the tritiated β-lysine obtained in such experiments yielded a sample of tritiated succinic acid which was shown in an enzymic assay involving partial oxidation with succinate dehydrogenase, to possess the (S)-configuration. Thus, the overall substitution at C(5) occurs with inversion of configuration.     

Vitamin B12-derivatives-enzyme cofactors and ligands of proteins and nucleic acids

B(12)-cofactors play important roles in the metabolism of microorganisms, animals and humans. Microorganisms are the only natural sources of B(12)-derivatives, and the latter are "vitamins" for other B(12)-requiring organisms. Some B(12)-dependent enzymes catalyze complex isomerisation reactions, such as methylmalonyl-CoA mutase. They need coenzyme B(12), an organometallic B(12)-derivative, to induce enzymatic radical reactions. Another group of widely relevant enzymes catalyzes the transfer of methyl groups, such as methionine synthase, which uses methylcobalamin as cofactor. This tutorial review covers structure and reactivity of B(12)-derivatives and structural aspects of their interactions with proteins and nucleotides, which are crucial for the efficient catalysis by the important B(12)-dependent enzymes, and for achieving and regulating uptake and transport of B(12)-derivatives.     

Molecular evolution of B<Subscript>6</Subscript> enzymes: Binding of pyridoxal-5'-phosphate and Lys41Arg substitution turn ribonuclease A into a model B<Subscript>6</Subscript> protoenzyme

Background  

The pyridoxal-5'-phosphate (PLP)-dependent or vitamin B₆-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids belong to no fewer than four evolutionarily independent protein families. The multiple evolutionary origin and the essential mechanistic role of PLP in these enzymes argue for the cofactor having arrived on the evolutionary scene before the emergence of the respective apoenzymes and having played a dominant role in the molecular evolution of the B₆ enzyme families. Here we report on an attempt to re-enact the emergence of a PLP-dependent protoenzyme. The starting protein was pancreatic ribonuclease A (RNase), in which active-site Lys41 or Lys7 readily form a covalent adduct with PLP.     

Glycerol Dehydratases: Biochemical Structures,Catalytic Mechanisms,and Industrial Applications in 1,3-Propanediol Production by Naturally Occurring and Genetically Engineered Bacterial Strains

To date, two types of glycerol dehydratases have been reported: coenzyme B₁₂-dependent and coenzyme B₁₂-independent glycerol dehydratases. The three-dimensional structure of the former is a dimer of αβγ heterotrimer, while that of the latter is a homodimer. Their mechanisms of reaction are typically enzymatic radical catalysis. Functional radical in both the glycerol dehydratases is the adenosyl radical. However, the adenosyl radical in the former originates from coenzyme B₁₂ by homolytic cleavage, and that in the latter from S-adenosyl-methionine. Until some years ago, Clostridium butyricum VPI 1718 was the only microorganism known to possess B₁₂-independent glycerol dehydratase, but since then, several other bacteria with this unique capability have been identified. This article focuses on the glycerol dehydratases and on 1,3-propanediol production from glycerol by naturally occurring and genetically engineered bacterial strains containing glycerol dehydratase.     

Glycerol as a Substrate and Inactivator of Coenzyme B12-Dependent Diol Dehydratase

Diol dehydratase, dependent on coenzyme B₁₂ (B₁₂-dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B₁₂-independent glycerol dehydratase (B₁₂-iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B₁₂-dDDH and that of resistance of B₁₂-iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B₁₂-dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B₁₂-iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.     

Superelectrophilic Behavior of an Anion Demonstrated by the Spontaneous Binding of Noble Gases to [B12Cl11]−

It is common and chemically intuitive to assign cations electrophilic and anions nucleophilic reactivity, respectively. Herein, we demonstrate a striking violation of this concept: The anion [B₁₂Cl₁₁]⁻ spontaneously binds to the noble gases (Ngs) xenon and krypton at room temperature in a reaction that is typical of “superelectrophilic” dications. [B₁₂Cl₁₁Ng]⁻ adducts, with Ng binding energies of 80 to 100 kJ mol⁻¹, contain B−Ng bonds with a substantial degree of covalent interaction. The electrophilic nature of the [B₁₂Cl₁₁]⁻ anion is confirmed spectroscopically by the observation of a blue shift of the CO stretching mode in the IR spectrum of [B₁₂Cl₁₁CO]⁻ and theoretically by investigation of its electronic structure. The orientation of the electric field at the reactive site of [B₁₂Cl₁₁]⁻ results in an energy barrier for the approach of polar molecules and facilitates the formation of Ng adducts that are not detected with reactive cations such as [C₆H₅]⁺. This introduces the new chemical concept of “dipole-discriminating electrophilic anions.”     

Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 and factor IV

Background: In order to study the biosynthesis of vitamin B₁₂, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates.Results: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-l-methionine were included in the incubation, the product was precorrin-4. Both precorrin-3B and precorrin-4 are known precursors of vitamin B₁₂ and their availability has allowed new mechanistic studies of enzymic transformations.Conclusions: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B₁₂. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B₁₂ biosynthesis.     

Methyl transfer from a hydrophobic vitamin B12 derivative to arsenic trioxide

The methylation reaction of arsenic trioxide conducted at 37 °C and pH 7.0 for 24 h using hydrophobic methylated vitamin B₁₂, (methyl) (aquo) heptamethylcobyrinate perchlorate, CH₃B₁₂ ester, as a methyl donor in the presence of reduced glutathione (GSH) yielded monomethylarsonous acid (MMA), dimethylarsinic acid (DMA), and trimethylarsine oxide (TMAO) as products with a methylation rate over 95%. In contrast, when methylcobalamin (CH₃B₁₂) was used as the methyl donor, only MMA and DMA were produced and the methylation rate dropped to around 20%. Reductive demethylation of a methyl-corrinoid coordination complex mediated by GSH is suggested as a mechanism of methyl transfer to arsenic trioxide. The differences observed for different corrinoid coordination complexes with respect to the reactivity of methyl transfer to arsenic is ascribable to differences inherent in the base-on (CH₃B₁₂) and base-off (CH₃B₁₂ ester) natures of the compounds.     

Enzymatic radical catalysis: coenzyme B12-dependent diol dehydratase

A peculiar function resides in a peculiar structure. Coenzyme B₁₂ or adenosylcobalamin, a naturally occurring organometallic compound, serves as a cofactor for enzymatic radical reactions. How do the enzymes form catalytic radicals at the active sites? How do the enzymes utilize and control the high reactivity of the radicals for catalysis? Recently, three‐dimensional structures of several radical‐containing or radical‐forming enzymes including B₁₂ enzymes have been reported, enabling the analysis of the fine mechanisms of the action of these interesting enzymes. Our biochemical, mutational, and crystallographic studies as well as theoretical calculations on diol dehydratase, an adenosylcobalamin–dependent enzyme, revealed that its structure is adapted for its function—that is, activation of the Co? C bond toward homolysis, abstraction of a specific hydrogen atom from the substrate and its recombination to a particular product, and transition state stabilization in the hydroxyl group migration of a substrate‐derived radical. The functions of K⁺ and the active‐site amino acid residues in enzyme catalysis are also investigated. Based on the results, the fine mechanism of the enzyme and the energetic feasibility of enzymatic radical catalysis are described here. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 2: 352–366, 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10035     

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.     

Antivitamins B12—Some Inaugural Milestones

The recently delineated structure- and reactivity-based concept of antivitamins B₁₂ has begun to bear fruit by the generation, and study, of a range of such B₁₂-dummies, either vitamin B₁₂-derived, or transition metal analogues that also represent potential antivitamins B₁₂ or specific B₁₂-antimetabolites. As reviewed here, this has opened up new research avenues in organometallic B₁₂-chemistry and bioinorganic coordination chemistry. Exploratory studies with antivitamins B₁₂ have, furthermore, revealed some of their potential, as pharmacologically interesting compounds, for inducing B₁₂-deficiency in a range of organisms, from hospital resistant bacteria to laboratory mice. The derived capacity of antivitamins B₁₂ to induce functional B₁₂-deficiency in mammalian cells and organs also suggest their valuable potential as growth inhibitors of cancerous human and animal cells.     

Komplexbildung des Hydroxycobalamins mit Kaliumhexacyanoferrat(II)

Zusammenfassung Das Hydroxycobalamin bildet mit Kaliumhexacyanoferrat(II) bei pH 2,5–2,7 ein wasserlösliches Derivat [B₁₂-Fe(CN)₆-B₁₂]^2–, das einen Komplex mit einer Bildungskonstante lgK=10,00±0,04 darstellt. Der Komplex ist reaktionsfähig und wird leicht in andere Derivate des Vitamins B₁₂ übergeführt.

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Complex Formation of hydroxycobalamin with potassium hexacyanoferrate(II)

Hydroxycobalamin forms with potassium hexacyanoferrate(II) at pH 2.5–2.7 a water soluble complex [B₁₂-Fe(CN)₆-B₁₂]^2– with a formation constant lgK=10.00±0.04. The complex has high reactivity and easily can be converted into other derivatives of vitamin B₁₂.

     

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.     

Probing Reactivity of PQQ-Dependent Carbohydrate Dehydrogenases Using Artificial Electron Acceptor

The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were determined using various electron acceptors. The radical cations of organic compounds and 2,6-dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose. The reactivity of dioxygen with ASDH is low; the bimolecular constant k _ox = 660 M⁻¹ s⁻¹, while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of dehydrogenases carbohydrates specificity. Mono- and disaccharide reactivity with GDH is higher than the reactivity of oligosaccharides. For ASDH, the reactivity increased with the carbohydrate monomer number increase. The specificity of quinoproteins was compared with specificity of flavoprotein Microdochium nivale carbohydrate oxidase due to potential enzymes application for lactose oxidation.