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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.

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.

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

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.

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.

Thiaminases are responsible for the degradation of thiamin and its metabolites. Two classes of thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. Although the reactions of several thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. We have determined the three-dimensional X-ray structure of an inactive C143S mutant of Clostridium botulinum (Cb) thiaminase I with bound thiamin at 2.2 Å resolution. The C143S/thiamin complex provides atomic level details of the orientation of thiamin upon binding to Cb-thiaminase I and the identity of active site residues involved in substrate binding and catalysis. The specific roles of active site residues were probed by using site directed mutagenesis and kinetic analyses, leading to a detailed mechanism for Cb-thiaminase I. The structure of Cb-thiaminase I is also compared to the functionally similar but structurally distinct thiaminase II.

HpxO is a flavin-dependent urate oxidase that catalyzes the hydroxylation of uric acid to 5-hydroxyisourate and functions in a novel pathway for purine catabolism found in Klebsiella pneumoniae. We have determined the structures of HpxO with and without uric acid at 2.0 and 2.2 A, respectively. We have also determined the structure of the R204Q variant at 2.0 A resolution in the absence of uric acid. The variant structure is very similar to that of wild-type HpxO except for the conformation of Arg103, which interacts with FAD in the variant but not in the wild-type structure. Interestingly, the R204Q variant results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation. This suggests that Arg204 facilitates the deprotonation of uric acid, activating it for the oxygen transfer. On the basis of these data, a mechanism for this reaction consisting of a nucleophilic attack of the urate anion on the flavin hydroperoxide resulting in the formation of 5-hydroxyisourate is ...

Thiamin diphosphate is an essential cofactor in all forms of life and plays a key role in amino acid and carbohydrate metabolism. Its biosynthesis involves separate syntheses of the pyrimidine and thiazole moieties, which are then coupled to form thiamin monophosphate. A final phosphorylation produces the active form of the cofactor. In most bacteria, six gene products are required for biosynthesis of the thiamin thiazole. In yeast and fungi only one gene product, Thi4, is required for thiazole biosynthesis. Methanococcus jannaschii expresses a putative Thi4 ortholog that was previously reported to be a ribulose 1,5-bisphosphate synthase [Finn, M. W. and Tabita, F. R. (2004) J. Bacteriol., 186, 6360-6366]. Our structural studies show that the Thi4 orthologs from M. jannaschii and Methanococcus igneus are structurally similar to Thi4 from Saccharomyces cerevisiae. In addition, all active site residues are conserved except for a key cysteine residue, which in S. cerevisiae is the source of the thiazole sulfur atom. Our recent biochemical studies showed that the archael Thi4 orthologs use nicotinamide adenine dinucleotide, glycine, and free sulfide to form the thiamin thiazole in an iron-dependent reaction [Eser, B., Zhang, X., Chanani, P. K., Begley, T. P., and Ealick, S. E. (2016) J. Am. Chem. Soc. , DOI: 10.1021/jacs.6b00445]. Here we report X-ray crystal structures of Thi4 from M. jannaschii complexed with ADP-ribulose, the C205S variant of Thi4 from S. cerevisiae with a bound glycine imine intermediate, and Thi4 from M. igneus with bound glycine imine intermediate and iron. These studies reveal the structural basis for the iron-dependent mechanism of sulfur transfer in archael and yeast thiazole synthases.

Pseudouridine (Ψ) was the first modified nucleoside to be discovered and is the most abundant modification in RNA, existing in tRNAs, rRNAs, snRNAs and snoRNAs (1). Ψ is biosynthesized post-transcriptionally from RNA uridine moieties by Ψ synthase, which cleaves the glycosidic C-N bond and reconnects the uracil to the ribosyl moiety at the C5 position (2). Pseudouridine reduces the conformational flexibility of RNA because the exposed, protonated N1 atom forms strong hydrogen bonds with structured water molecules, replacing weak interactions formed by C5 (1, 3). While Ψ biosynthesis has been studied extensively, little is known about Ψ catabolism. Recently, the two enzymes responsible for Ψ 1 degradation in Escherichia coli were identified (4). Ψ kinase first phosphorylates Ψ to pseudouridine 5′-phosphate (ΨMP, 2). ΨMP glycosidase then catalyzes the reversible cleavage of the C-C glycosidic bond to form uracil 4 and ribose 5-phosphate (R5P, 3) (Scheme 1). While ΨMP glycosidase functions biologically in the cleavage direction, equilibrium strongly favors ΨMP synthesis with an equilibrium constant for the degradation reaction of 2.3 × 10-4 M (4). In bacterial genomes Ψ kinase and ΨMP glycosidase are encoded by separate genes. In E. coli these genes are yeiC and yeiN, respectively, which are located in the same operon. In some eukaryotes, the genes for Ψ kinase and ΨMP glycosidase are fused, resulting in a bifunctional enzyme. Enzymes metabolizing Ψ are not found in humans and most other higher organisms, and excess Ψ is excreted (5). Scheme 1 While natural products containing O and N glycosidic bonds are widespread, natural products containing the C-glycosidic bond are much less prevalent (6). The C-glycosidic bond is generally assumed to be formed by an electrophilic aromatic substitution. Enzymes that cleave C-N glycosidic bonds have been extensively studied. These include nucleoside and nucleotide hydrolases (7-9), purine and pyrimidine nucleoside phosphorylases (10), purine and pyrimidine phosphoribosyltransferases (11, 12), and nucleoside deoxyribosyltransferases (13). In addition, DNA-repair enzymes such as endonuclease III function as N-glycosylases to excise damaged nucleobases from DNA (14). Enzymes that cleave C-O glycosidic bonds are widespread in carbohydrate metabolism and have also been extensively studied (15). Biochemical, biophysical and genetic studies suggest that these enzymes cleave C-N and C-O glycosidic bonds by a largely dissociative mechanism generating an oxocarbenium ion intermediate. While the genes encoding several C-glycosynthases have been identified and in many cases overexpressed (16-23) – and the structures of ligand free UrdGT2 (24, 25) and ΨMP glycosidase (26) have been determined – there is currently no example of a mechanistically well-characterized C-glycosidase or glycosynthase. Here we report a set of E. coli ΨMP glycosidase (EcΨMP glycosidase) crystal structures, which provide four snapshots of the reaction coordinate. The structures are: the unliganded enzyme, a ΨMP glycosidase/ring-opened R5P adduct, a ΨMP glycosidase/ ring-opened ribose ΨMP adduct, and a K166A ΨMP complex. The structural studies suggested that the reaction involves an intermediate imine with Lys166. This was confirmed by mass spectrometry. Kinetic studies on active site mutants suggested roles for individual amino acid side chains. Comparison of the EcΨMP glycosidase structures suggested a role for conformational changes in ΨMP cleavage and product release. These results suggest an unanticipated mechanism for the pseudouridine glycosidase catalyzed C-glycosyl bond hydrolysis.

High resolution crystal structures are reported for apo, holo, and substrate-bound forms of a toxoflavin-degrading metalloenzyme (TflA). In addition, the degradation reaction is shown to be dependent on oxygen, Mn(II), and dithiothreitol in vitro. Despite its low sequence identity with proteins of known structure, TflA is structurally homologous to proteins of the vicinal oxygen chelate superfamily. Like other metalloenzymes in this superfamily, the TflA fold contains four modules that associate to form a metal binding site; however, the fold displays a rare rearrangement of the structural modules indicative of domain permutation. Moreover, unlike the 2-His-1-carboxylate facial triad commonly utilized by vicinal oxygen chelate dioxygenases and other dioxygen-activating non-heme Fe(II) enzymes, the metal center in TflA consists of a 1-His-2-carboxylate facial triad. The substrate-bound complex shows square-pyramidal geometry in which one position is occupied by O5 of toxoflavin. The open coordination site is predicted to be the dioxygen binding site. TflA appears to stabilize the reduced form of toxoflavin through second-sphere interactions. This anionic species is predicted to be the electron source responsible for reductive activation of oxygen to produce a peroxytoxoflavin intermediate.

Bacimethrin-derived 2′-methoxythiamin pyrophosphate inhibits microbial growth by disrupting metabolic pathways dependent on thiamin-utilizing enzymes. This study describes the discovery of the bacimethrin biosynthetic gene cluster of Clostridium botulinum A ATCC 19397 and in vitro reconstitution of bacimethrin biosynthesis from cytidine 5′-monophosphate.