The structural and biochemical foundations of thiamin biosynthesis.

Thiamin is synthesized by most prokaryotes and by eukaryotes such as yeast and plants. In all cases, the thiazole and pyrimidine moieties are synthesized in separate branches of the pathway and coupled to form thiamin phosphate. A final phosphorylation gives thiamin pyrophosphate, the active form of the cofactor. Over the past decade or so, biochemical and structural studies have elucidated most of the details of the thiamin biosynthetic pathway in bacteria. Formation of the thiazole requires six gene products, and formation of the pyrimidine requires two. In contrast, details of the thiamin biosynthetic pathway in yeast are only just beginning to emerge. Only one gene product is required for the biosynthesis of the thiazole and one for the biosynthesis of the pyrimidine. Thiamin can also be transported into the cell and can be salvaged through several routes. In addition, two thiamin degrading enzymes have been characterized, one of which is linked to a novel salvage pathway.

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.

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.

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.

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.].

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.

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.

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.

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.

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.

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand ß6 of the Pdx1 (ßα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.

Mechanistic Studies on Tryptophan Lyase (NosL): Identification of Cyanide as a Reaction Product.

Tryptophan lyase (NosL) catalyzes the formation of 3-methylindole-2-carboxylic acid and 3-methylindole from l-tryptophan. In this paper, we provide evidence supporting a formate radical intermediate and demonstrate that cyanide is a byproduct of the NosL-catalyzed reaction with l-tryptophan. These experiments require a major revision of the NosL mechanism and uncover an unanticipated connection between NosL and HydG, the radical SAM enzyme that forms cyanide and carbon monoxide from tyrosine during the biosynthesis of the metallo-cluster of the [Fe-Fe] hydrogenase.

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.

Anaerobic biosynthesis of the lower ligand of vitamin B12.

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 intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B12 itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.

RutA-Catalyzed Oxidative Cleavage of the Uracil Amide Involves Formation of a Flavin-N5-oxide.

RutA is a novel flavoenzyme on the uracil catabolic pathway that catalyzes uracil ring opening by a unique amide oxidation reaction. Here we provide evidence that this reaction also involves the formation of a flavin-N5-oxide.

Biochemical Characterization and Structural Basis of Reactivity and Regioselectivity Differences between Burkholderia thailandensis and Burkholderia glumae 1,6-Didesmethyltoxoflavin N-Methyltransferase.

Burkholderia glumae converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to BgToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B. thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase that shows a low level of sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a class I methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine ring system is orthogonal to its orientation in BgToxA. In BthII1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in BgToxA, it is first directed toward an sp2 orbital of N6 and then toward an sp2 orbital of N1 after planar rotation of the azapteridine ring system. Furthermore, in BthII1283, N1 is hydrogen bonded to a histidine residue whereas BgToxA does not supply an obvious basic residue for either N6 or N1 methylation.

Identification of the First Riboflavin Catabolic Gene Cluster Isolated from Microbacterium maritypicum G10.

Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed.