Radical S-Adenosylmethionine (SAM) Enzymes in Cofactor Biosynthesis: A Treasure Trove of Complex Organic Radical Rearrangement Reactions (original) (raw)
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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.
S-Adenosylmethionine radical enzymes
Bioorganic Chemistry, 2004
The role of S-adenosylmethionine (SAM) as a precursor to organic radicals, generated by one-electron reduction of SAM and subsequent Wssion to form 5Ј-deoxyadenosyl radical and methionine, has been known for some time. Only recently, however, has it become apparent how widespread such enzymes are, and what a wide range of chemical reactions they catalyze. In the last few years several new SAM radical enzymes have been identiWed. Spectroscopic and kinetic investigations have begun to uncover the mechanism by which an iron sulfur cluster unique to these enzymes reduces SAM to generate adenosyl radical. Most recently, the Wrst Xray structures of SAM radical enzymes, coproporphyrinogen-III oxidase, and biotin synthase have been solved, providing a structural framework within which to interpret mechanistic studies. 2004 Elsevier Inc. All rights reserved.
The generation of 5′-deoxyadenosyl radicals by adenosylmethionine-dependent radical enzymes
Current Opinion in Chemical Biology, 2003
Adenosylmethionine-dependent radical enzymes provide a novel mechanism for generating the highly oxidizing 5 0 -deoxyadenosyl radical in an anaerobic reducing environment. Recent studies suggest a unique covalent interaction between adenosylmethionine and a catalytic iron-sulfur cluster that may promote inner-sphere electron transfer to the sulfonium, resulting in the reductive cleavage of a C-S bond and the generation of a 5 0 -deoxyadenosyl radical. The utilization of this radical as a catalytic and stoichiometric oxidant in many different enzyme reactions reflects the broad diversity of radical enzymes throughout biology.
Nucleic Acids Research, 2001
A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously wellstudied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.
Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2012
Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The π-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
Biotin synthase: Insights into radical-mediated carbon–sulfur bond formation
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2012
The enzyme cofactor and essential vitamin biotin is biosynthesized in bacteria, fungi, and plants through a pathway that culminates with the addition of a sulfur atom to generate the five-membered thiophane ring. The immediate precursor, dethiobiotin, has methylene and methyl groups at the C6 and C9 positions, respectively, and formation of a thioether bridging these carbon atoms requires cleavage of unactivated C\H bonds. Biotin synthase is an S-adenosyl-L-methionine (SAM or AdoMet) radical enzyme that catalyzes reduction of the AdoMet sulfonium to produce 5′-deoxyadenosyl radicals, high-energy carbon radicals that can directly abstract hydrogen atoms from dethiobiotin. The available experimental and structural data suggest that a [2Fe-2S] 2 + cluster bound deep within biotin synthase provides a sulfur atom that is added to dethiobiotin in a stepwise reaction, first at the C9 position to generate 9-mercaptodethiobiotin, and then at the C6 position to close the thiophane ring. The formation of sulfur-containing biomolecules through a radical reaction involving an iron-sulfur cluster is an unprecedented reaction in biochemistry; however, recent enzyme discoveries suggest that radical sulfur insertion reactions may be a distinct subgroup within the burgeoning Radical SAM superfamily. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
Carbon–sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE
Nature Chemistry, 2016
Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5′-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.
Radical Breakthroughs in Natural Product and Cofactor Biosynthesis
Biochemistry, 2018
The radical SAM (S-adenosyl-l-methionine) superfamily is one of the largest group of enzymes with >113000 annotated sequences [Landgraf, B. J., et al. (2016) Annu. Rev. Biochem. 85, 485-514]. Members of this superfamily catalyze the reductive cleavage of SAM using an oxygen sensitive 4Fe-4S cluster to transiently generate 5'-deoxyadenosyl radical that is subsequently used to initiate diverse free radical-mediated reactions. Because of the unique reactivity of free radicals, radical SAM enzymes frequently catalyze chemically challenging reactions critical for the biosynthesis of unique structures of cofactors and natural products. In this Perspective, I will discuss the impact of characterizing novel functions in radical SAM enzymes on our understanding of biosynthetic pathways and use two recent examples from my own group with a particular emphasis on two radical SAM enzymes that are responsible for carbon skeleton formation during the biosynthesis of a cofactor and natural p...
Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase
Nature Communications, 2015
Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5 0-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a b-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the b-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered clusterbinding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.
S-Adenosylmethionine-dependent radical-based modification of biological macromolecules
Current Opinion in Structural Biology, 2010
Proteins and RNA molecules enjoy a variety of chemically complex post-translational and post-transcriptional modifications. The chemistry at work in these reactions, which was considered to be exclusively ionic in nature has recently been shown to depend on radical mechanisms in some cases. The overwhelming majority of these radical-based reactions are catalyzed by 'Radical-SAM' enzymes. This Scheme 1 Mechanism of substrate activation by Radical-SAM enzymes. S-H: substrate, tRNA, rRNA or protein; Ado : 5 0 -deoxyadenosyl radical; S : radical substrate; P: product; Met: methionine; [4Fe-4S]-SAM: [4Fe-4S] cluster in complex with SAM; SAM shown in red is a ligand to the fourth iron in red, cysteine residues of the CysX 3 CysX 2 Cys motif are shown in green.