Phosphomethylpyrimidine Synthase (ThiC): Trapping of Five Intermediates Provides Mechanistic Insights on a Complex Radical Cascade Reaction in Thiamin Biosynthesis.

Phosphomethylpyrimidine synthase (ThiC) catalyzes the conversion of AIR to the thiamin pyrimidine HMP-P. This reaction is the most complex enzyme-catalyzed radical cascade identified to date, and the detailed mechanism has remained elusive. In this paper, we describe the trapping of five new intermediates that provide snapshots of the ThiC reaction coordinate and enable the formulation of a revised mechanism for the ThiC-catalyzed reaction.

Alkylcysteine Sulfoxide C-S Monooxygenase Uses a Flavin-Dependent Pummerer Rearrangement.

Flavoenzymes are highly versatile and participate in the catalysis of a wide range of reactions, including key reactions in the metabolism of sulfur-containing compounds. S-Alkyl cysteine is formed primarily by the degradation of S-alkyl glutathione generated during electrophile detoxification. A recently discovered S-alkyl cysteine salvage pathway uses two flavoenzymes (CmoO and CmoJ) to dealkylate this metabolite in soil bacteria. CmoO catalyzes a stereospecific sulfoxidation, and CmoJ catalyzes the cleavage of one of the sulfoxide C-S bonds in a new reaction of unknown mechanism. In this paper, we investigate the mechanism of CmoJ. We provide experimental evidence that eliminates carbanion and radical intermediates and conclude that the reaction proceeds via an unprecedented enzyme-mediated modified Pummerer rearrangement. The elucidation of the mechanism of CmoJ adds a new motif to the flavoenzymology of sulfur-containing natural products and demonstrates a new strategy for the enzyme-catalyzed cleavage of C-S bonds.

Oxidative Dearomatization of PLP in Thiamin Pyrimidine Biosynthesis in Candida albicans.

The yeast thiamin pyrimidine synthase THI5p catalyzes one of the most complex organic rearrangements found in primary metabolism. In this reaction, the active site His66 and PLP are converted to thiamin pyrimidine in the presence of Fe(II) and oxygen. The enzyme is a single-turnover enzyme. Here, we report the identification of an oxidatively dearomatized PLP intermediate. We utilize oxygen labeling studies, chemical-rescue-based partial reconstitution experiments, and chemical model studies to support this identification. In addition, we also identify and characterize three shunt products derived from the oxidatively dearomatized PLP.

Retraction notice to "Comparison of thiaminase activity in fish using the radiometric and 4-nitrothiophenol colorimetric methods" [J. Great Lakes Res. 36 (2010) 641-645].

[This retracts the article PMC6042866.].

Mechanistic Studies on the Single-Turnover Yeast Thiamin Pyrimidine Synthase: Characterization of the Inactive Enzyme.

The eukaryotic thiamin pyrimidine synthase, THI5p, has been identified as a suicidal/single-turnover enzyme that catalyzes the conversion of its active site histidine and lysine-bound pyridoxal phosphate (PLP) to the thiamin pyrimidine (HMP-P). Here we identify the histidine and PLP fragments using bottom-up proteomics and LC-MS analysis. We also identify the active form of the iron cofactor and quantitate the oxygen requirement of the THI5p reaction. This information is integrated into a mechanistic proposal for this remarkable reaction.

Cysteine Dealkylation in Bacillus subtilis by a Novel Flavin-Dependent Monooxygenase.

In this paper, we describe the biochemical reconstitution of a cysteine salvage pathway and the biochemical characterization of each of the five enzymes involved. The salvage begins with amine acetylation of S-alkylcysteine, followed by thioether oxidation. The C-S bond of the resulting sulfoxide is cleaved using a new flavoenzyme catalytic motif to give N-acetylcysteine sulfenic acid. This is then reduced to the thiol and deacetylated to complete the salvage pathway. We propose that this pathway is important in the catabolism of alkylated cysteine generated by proteolysis of alkylated glutathione formed in the detoxification of a wide range of electrophiles.

Trapping and structural characterisation of a covalent intermediate in vitamin B6 biosynthesis catalysed by the Pdx1 PLP synthase.

The Pdx1 enzyme catalyses condensation of two carbohydrates and ammonia to form pyridoxal 5-phosphate (PLP) via an imine relay mechanism of carbonyl intermediates. The I333 intermediate characterised here using structural, UV-vis absorption spectroscopy and mass spectrometry analyses rationalises stereoselective deprotonation and subsequent substrate assisted phosphate elimination, central to PLP biosynthesis.

Radiosynthesis of 5-[18F]Fluoro-1,2,3-triazoles through Aqueous Iodine-[18F]Fluorine Exchange Reaction.

In this report, a simple and efficient process to achieve fluorine-18-labeled 1,2,3-triazole is reported. The heteroaromatic radiofluorination was successfully achieved through an iodine-fluorine-18 exchange in an aqueous medium requiring only trace amounts of base and no azeotropic drying of fluorine-18. This methodology was optimized on a model reaction and further validated on multiple 1,2,3-triazole substrates with 18-60% radiochemical conversions. Using this strategy-the radiosynthesis of a triazole-based thiamin analogue-a potential positron emission tomography (PET) probe for imaging thiamin-dependent enzymes was synthesized with 10-16% isolated radiochemical yield (RCY) in 40 min (uncorrected, n > 5).

Menaquinone Biosynthesis: The Mechanism of 5,8-Dihydroxy-2-naphthoate Synthase (MqnD).

MqnD catalyzes the conversion of cyclic dehypoxanthine futalosine (6) to 5,8-dihydroxy-2-naphthoic acid (7) and an uncharacterized product. This study describes a chemoenzymatic synthesis of 6. This synthesis achieved a 2-fold yield enhancement by using titanium(III) citrate as the reducing agent and another 5-fold yield enhancement using a fluorinated analogue of dehypoxanthine futalosine (5) that was converted to 6 by an ipso substitution mechanism. This synthetic route enabled the synthesis of 6 in sufficient quantity to identify the second reaction product and to determine that the MqnD-catalyzed reaction proceeds by a hemiacetal ring opening-tautomerization-retroaldol sequence.

Menaquinone Biosynthesis: New Strategies to Trap Radical Intermediates in the MqnE-Catalyzed Reaction.

Aminofutalosine synthase (MqnE) is a radical SAM enzyme that catalyzes the conversion of 3-((1-carboxyvinyl)oxy)benzoic acid to aminofutalosine during the futalosine-dependent menaquinone biosynthesis. In this Communication, we report the trapping of a radical intermediate in the MqnE-catalyzed reaction using sodium dithionite, molecular oxygen, or 5,5-dimethyl-1-pyrroline-N-oxide. These radical trapping strategies are potentially of general utility in the study of other radical SAM enzymes.

Structural basis for antibiotic action of the B1 antivitamin 2'-methoxy-thiamine.

The natural antivitamin 2'-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2'-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential.

Radical SAM Enzyme HydE Generates Adenosylated Fe(I) Intermediates En Route to the [FeFe]-Hydrogenase Catalytic H-Cluster.

The H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S]H-subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe]H-subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe]H-subcluster is assembled by a set of Fe-S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [FeII(Cys)(CO)2(CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5'-deoxyadenosyl radical attacking the cysteine S to form a C5'-S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3-S bond, producing a mononuclear [FeI(CO)2(CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe]H-subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.

Mechanistic Studies on CysS - A Vitamin B12-Dependent Radical SAM Methyltransferase Involved in the Biosynthesis of the tert-Butyl Group of Cystobactamid.

Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) methyltransferases catalyze methylation reactions at non-nucleophilic centers in a wide range of substrates. CysS is a Cbl-dependent radical SAM methyltransferase involved in cystobactamid biosynthesis. This enzyme catalyzes the sequential methylation of a methoxy group to form ethoxy, i-propoxy, s-butoxy, and t-butoxy groups on a p-aminobenzoate peptidyl carrier protein thioester intermediate. This biosynthetic strategy enables the host myxobacterium to biosynthesize a combinatorial antibiotic library of 25 cystobactamid analogues. In this Article, we describe three experiments to elucidate how CysS uses Cbl, SAM, and a [4Fe-4S] cluster to catalyze iterative methylation reactions: a cyclopropylcarbinyl rearrangement was used to trap the substrate radical and to estimate the rate of the radical substitution reaction involved in the methyl transfer; a bromoethoxy analogue was used to explore the active site topography; and deuterium isotope effects on the hydrogen atom abstraction by the adenosyl radical were used to investigate the kinetic significance of the hydrogen atom abstraction. On the basis of these experiments, a revised mechanism for CysS is proposed.

Trapping and Electron Paramagnetic Resonance Characterization of the 5'dAdo• Radical in a Radical S-Adenosyl Methionine Enzyme Reaction with a Non-Native Substrate.

S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S-adenosyl methionine to generate the 5'-deoxyadenosyl radical (5'dAdo•), a potent H-atom abstractor. HydG, a member of this rSAM family, uses the 5'dAdo• radical to lyse its substrate, tyrosine, producing CO and CN that bind to a unique Fe site of a second HydG Fe-S cluster, ultimately producing a mononuclear organometallic Fe-l-cysteine-(CO)2CN complex as an intermediate in the bioassembly of the catalytic H-cluster of [Fe-Fe] hydrogenase. Here we report the use of non-native tyrosine substrate analogues to further probe the initial radical chemistry of HydG. One such non-native substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the amino group of tyrosine, replacing the CαH-NH2 with a CH2 at the C2 position. Electron paramagnetic resonance (EPR) studies show the generation of a strong and relatively stable radical in the HydG reaction with natural abundance and 13C2-HPPA, with appreciable spin density localized at C2. These results led us to try parallel experiments with the more oxidized non-native substrate coumaric acid, which has a C2=C3 alkene substitution relative to HPPA's single bond. Interestingly, the HydG reaction with the cis-p-coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using cis-p-coumaric acid along with isotopically labeled SAM reveal that we have for the first time trapped and characterized the 5'dAdo• radical in an actual rSAM enzyme reaction, here by using this specific non-native substrate cis-p-coumaric acid. Density functional theory energetics calculations show that the cis-p-coumaric acid has approximately the same C-H bond dissociation free energy as 5'dAdo•, providing a possible explanation for our ability to trap an appreciable fraction of 5'dAdo• in this specific rSAM reaction. The radical's EPR line shape and its changes with SAM isotopic substitution are nearly identical to those of a 5'dAdo• radical recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM center in another rSAM enzyme, pyruvate formate-lyase activating enzyme, further supporting our assignment that we have indeed trapped and characterized the 5'dAdo• radical in a radical SAM enzymatic reaction by appropriate tuning of the relative radical free energies via the judicious selection of a non-native substrate.

Flavin-N5-oxide intermediates in dibenzothiophene, uracil, and hexachlorobenzene catabolism.

Flavin-N5-oxide is a new intermediate in flavoenzymology. Here we describe the identification of DszA (dibenzothiophene catabolism), RutA (uracil catabolism) and HcbA1 (hexachlorobenzene catabolism) as flavin-N5-oxide-utilizing enzymes. Mechanistic analysis of these reactions suggests a model for the identification of other examples of this catalytic motif.

Menaquinone Biosynthesis: Biochemical and Structural Studies of Chorismate Dehydratase.

Menaquinone (MK, vitamin K) is a lipid-soluble quinone that participates in the bacterial electron transport chain. In mammalian cells, vitamin K functions as an essential vitamin for the activation of several proteins involved in blood clotting and bone metabolism. MqnA is the first enzyme on the futalosine-dependent pathway to menaquinone and catalyzes the aromatization of chorismate by water loss. Here we report biochemical and structural studies of MqnA. These studies suggest that the dehydration reaction proceeds by a variant of the E1cb mechanism in which deprotonation is slower than water loss and that the enol carboxylate of the substrate is serving as the base.

Antibacterial Strategy against H. pylori: Inhibition of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis.

Aminofutalosine synthase (MqnE) catalyzes an important rearrangement reaction in menaquinone biosynthesis by the futalosine pathway. In this Letter, we report the identification of previously unreported inhibitors of MqnE using a mechanism-guided approach. The best inhibitor shows efficient inhibitory activity against H. pylori (IC50 = 1.8 ± 0.4 µM) and identifies MqnE as a promising target for antibiotic development.

Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation.

HcbA1 is a unique flavoenzyme that catalyzes the first step in the bacterial hexachlorobenzene catabolic pathway. Here we report in vitro reconstitution of the HcbA1-catalyzed reaction. Detailed mechanistic studies provide evidence for nucleophilic aromatic substitution and flavin-N5-oxide formation.

Novel enzymology in futalosine-dependent menaquinone biosynthesis.

The recently discovered futalosine-dependent menaquinone biosynthesis pathway employs radical chemistry for the naphthoquinol core assembly. Mechanistic studies on this pathway have resulted in the discovery of novel reaction motifs. MqnA is the first example of a chorismate dehydratase. MqnE is the first example of a radical SAM enzyme that catalyzes the addition of the 5'-deoxyadenosyl radical to the substrate double bond rather than hydrogen atom abstraction. Both MqnE and MqnC reaction sequences involve radical additions to a benzene ring followed by formation of an aryl radical anion intermediate. The enzymology of the tailoring reactions after dihydroxynaphthoic acid formation remains to be elucidated. Since the futalosine-dependent menaquinone biosynthesis pathway is absent in humans, mechanistic studies on this pathway may promote the development of new antibiotics.

An Aminoimidazole Radical Intermediate in the Anaerobic Biosynthesis of the 5,6-Dimethylbenzimidazole Ligand to Vitamin B12.

Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathways depending on the presence of oxygen in the environment. The anaerobic biosynthesis pathway of 5,6-dimethylbenzimidazole, the so-called "lower ligand" to the cobalt center, has been recently identified. This process begins with the conversion of 5-aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (HBI) by the radical S-adenosyl-l-methionine (SAM) enzyme BzaF, also known as HBI synthase. In this work we report the characterization of a radical intermediate in the reaction of BzaF using electron paramagnetic resonance spectroscopy. Using various isotopologues of AIR, we extracted hyperfine parameters for a number of nuclei, allowing us to propose plausible chemical compositions and structures for this intermediate. Specifically, we find that an aminoimidazole radical is formed in close proximity to a fragment of the ribose ring. These findings induce the revision of past proposed mechanisms and illustrate the ability of radical SAM enzymes to tightly control the radical chemistry that they engender.