An Aminoimidazole Radical Intermediate in the Anaerobic Biosynthesis of the 5,6-Dimethylbenzimidazole Ligand to Vitamin B12 (original) (raw)
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Journal of the American Chemical Society, 2015
Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralog of thiC (thiC-1) clustered with anaerobic vitamin B12 biosynthetic genes. Here we demonstrate that ThiC-1 is a radical S-adenosylmethionine enzyme that catalyzes the remarkable conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI). We identify the origin of key product atoms and propose a reaction mechanism. These studies represent the first step in solving a long-standing problem in anaerobic vitamin B12 assembly and reveal an unanticipated intersection of thiamin and vitamin B12 biosynthesis.
2015
This dissertation focuses on radical S-adenosylmethionine enzymes involved in cofactor biosynthesis. Mechanistic studies discussed here include: (i) molybdenum cofactor biosynthetic enzyme-MoaA, (ii) thiamin pyrimidine synthase-ThiC (iii) hydroxybenzimidazole synthase, HBI synthase, involved in anaerobic vitamin B 12 biosynthesis. MoaA catalyzes the first step in molydopterin biosynthesis where GTP is converted to pterin. This catalysis involves a remarkable rearrangement reaction where the C8 of guanosine-5'-triphosphate (GTP) is inserted between the C2' and C3' carbon atoms of GTP to give the final pterin. Mechanistic studies involved characterization of the products of the reaction, identification of the position of hydrogen atom abstraction by 5'-deoxyadenosyl radical and trapping of intermediates by using 2',3'-dideoxyGTP, 2'-deoxyGTP and 2'-chloroGTP as substrate analogs. Thiamin pyrimidine synthase, ThiC, catalyzes a complex rearrangement reaction involving the conversion of aminoimidazole ribotide (AIR) to thiamin pyrimidine (HMP-P). A hydrogen atom transfer from S-adenosylmethionine (AdoMet) to HMP-P was demonstrated. Also, the stereochemistry of this transfer was elucidated. Bioinformatics studies on ThiC revealed that a paralog of ThiC was clustered with vitamin B 12 biosynthetic genes in several anaerobic microorganisms. The gene responsible for the anaerobic vitamin B 12-benzimidazole biosynthesis was previously unknown. We demonstrate that the gene product of this ThiC paralog is a radical Siii adenosylmethionine enzyme. Remarkably it catalyzes the conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI) and formate, and Sadenosylmethionine to 5'-deoxyadenosine. We determine the hydrogen atom abstracted by 5'-deoxyadenosyl radical. We also performed carbon, nitrogen and hydrogen labeling studies and characterized the labeling pattern on 5-HBI. Based on these studies we propose a reaction mechanism of this remarkable conversion of AIR to 5-HBI.
Anaerobic biosynthesis of the lower ligand of vitamin B12
Proceedings of the National Academy of Sciences of the United States of America, 2015
Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are i...
Concerning the intermediacy of organic radicals in vitamin B12-dependent enzymic reactions
Philosophical Transactions of the Royal Society of London. B, Biological Sciences, 1985
The vitamin B12coenzyme adenosylcobalamin assists the enzymic catalysis of molecular rearrangements of the type in which the migrating group X can be OH, NH2or a suitable substituted carbon atom such as C (=CH2)CO2H. This paper discusses evidence for the participation of organic radicals as intermediates in these reactions. Theoretical and model studies supporting the intermediacy of radicals in the reactions catalysed by the enzymes diol dehydratase and α-methyleneglutarate mutase are described. For the model studies, alkyl radicals, alkylcobaloximes (alkyl represents, for example, ethoxycarbonyl substituted, but-3-enyl and cyclopropylmethyl) and also dihydroxyalkylcobalamins have been investigated. The Co-Cα-Cβangle of 125° in adenosylcobalamin is shown to be an ‘especial’ angle by analysis of the crystal structures ofR- andS-2,3-dihydroxypropylcobalamin.
Aerobic synthesis of vitamin B12: ring contraction and cobalt chelation
Biochemical Society Transactions, 2005
The aerobic biosynthetic pathway for vitamin B12 (cobalamin) biosynthesis is reviewed. Particular attention is focused on the ring contraction process, whereby an integral carbon atom of the tetrapyrrole-derived macrocycle is removed. Previous work had established that this chemically demanding step is facilitated by the action of a mono-oxygenase called CobG, which generates a hydroxy lactone intermediate. This mono-oxygenase contains both a non-haem iron and an Fe-S centre, but little information is known about its mechanism. Recent work has established that in bacteria such as Rhodobacter capsulatus, CobG is substituted by an isofunctional protein called CobZ. This protein has been shown to contain flavin, haem and Fe-S centres. A mechanism is proposed to explain the function of CobZ. Another interesting aspect of the aerobic cobalamin biosynthetic pathway is cobalt insertion, which displays some similarity to the process of magnesium chelation in chlorophyll synthesis. The genet...
Structural Basis for Adenosylcobalamin Activation in AdoCbl-Dependent Ribonucleotide Reductases
ACS Chemical Biology, 2010
Class II ribonucleotide reductases (RNR) catalyze the formation of an essential thiyl radical by homolytic cleavage of the Co-C bond in their adenosylcobalamin (AdoCbl) cofactor. Several mechanisms for the dramatic acceleration of Co-C bond cleavage in AdoCbl-dependent enzymes have been advanced, but no consensus yet exists. We present the structure of the class II RNR from Thermotoga maritima in three complexes: (i) with allosteric effector dTTP, substrate GDP, and AdoCbl; (ii) with dTTP and AdoCbl; (iii) with dTTP, GDP, and adenosine. Comparison of these structures gives the deepest structural insights so far into the mechanism of radical generation and transfer for AdoCbl-dependent RNR. AdoCbl binds to the active site pocket, shielding the substrate, transient 5=-deoxyadenosyl radical and nascent thiyl radical from solution. The e-propionamide side chain of AdoCbl forms hydrogen bonds directly to the ␣-phosphate group of the substrate. This interaction appears to cause a "locking-in" of the cofactor, and it is the first observation of a direct cofactor-substrate interaction in an AdoCbl-dependent enzyme. The structures support an ordered sequential reaction mechanism with release or relaxation of AdoCbl on each catalytic cycle. A conformational change of the AdoCbl adenosyl ribose is required to allow hydrogen transfer to the catalytic thiol group. Previously proposed mechanisms for radical transfer in B12dependent enzymes cannot fully explain the transfer in class II RNR, suggesting that it may form a separate class that differs from the well-characterized eliminases and mutases. ARTICLE www.acschemicalbiology.org
Anaerobic synthesis of vitamin B12: characterization of the early steps in the pathway
Biochemical Society Transactions, 2005
The anaerobic biosynthesis of vitamin B 12 is slowly being unravelled. Recent work has shown that the first committed step along the anaerobic route involves the sirohydrochlorin (chelation of cobalt into factor II). The following enzyme in the pathway, CbiL, methylates cobalt-factor II to give cobalt-factor III. Recent progress on the molecular characterization of this enzyme has given a greater insight into its mode of action and specificity. Structural studies are being used to provide insights into how aspects of this highly complex biosynthetic pathway may have evolved. Between cobalt-factor III and cobyrinic acid, only one further intermediate has been identified. A combination of molecular genetics, recombinant DNA technology and bioorganic chemistry has led to some recent advances in assigning functions to the enzymes of the anaerobic pathway.
Concerning the Intermediacy of Organic Radicals in Vitamin B$_ $-Dependent Enzymic Reactions
Philosophical Transactions of the Royal Society B: Biological Sciences, 1985
The vitamin B 12 coenzyme adenosylcobalamin assists the enzymic catalysis of molecular rearrangements of the type in which the migrating group X can be OH, NH 2 or a suitable substituted carbon atom such as C (=CH 2 )CO 2 H. This paper discusses evidence for the participation of organic radicals as intermediates in these reactions. Theoretical and model studies supporting the intermediacy of radicals in the reactions catalysed by the enzymes diol dehydratase and α-methyleneglutarate mutase are described. For the model studies, alkyl radicals, alkylcobaloximes (alkyl represents, for example, ethoxycarbonyl substituted, but-3-enyl and cyclopropylmethyl) and also dihydroxyalkylcobalamins have been investigated. The Co-C α -C β angle of 125° in adenosylcobalamin is shown to be an ‘especial’ angle by analysis of the crystal structures of R - and S -2,3-dihydroxypropylcobalamin.