In Vitro Production of Ergothioneine Isotopologues

Ergothioneine is an emerging component of the redox homeostasis system in human cells and in microbial pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei. The synthesis of stable isotope-labeled ergothioneine derivatives may provide important tools for deciphering the distribution, function, and metabolism of this compound in vivo. We describe a general protocol for the production of ergothioneine isotopologues with programmable 2 H, 15 N, 13 C, 34 S, and 33 S isotope labeling patterns. This enzyme-based approach makes efficient use of commercial isotope reagents and is also directly applicable to the synthesis of radio-isotopologues

Structural and Mechanistic Basis for Anaerobic Ergothioneine Biosynthesis

Ergothioneine is an emergent factor in cellular redox biochemistry in humans and pathogenic bacteria. Broad consensus has formed around the idea that ergothioneine protects cells against reactive oxygen species. The recent discovery that anaerobic microorganisms make the same metabolite using oxygen-independent chemistry indicates that ergothioneine also plays physiological roles under anoxic conditions. In this report, we describe the crystal structure of the anaerobic ergothioneine biosynthetic enzyme EanB from green sulfur bacterium Chlorobium limicola. This enzyme catalyzes the oxidative sulfurization of N-alpha-trimethyl histidine. On the basis of structural and kinetic evidence, we describe the catalytic mechanism of this unusual C-S bond-forming reaction. Significant active-site conservation among distant EanB homologues suggests that the oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria

Enzymatic strategies for carbon-sulfur bond formation in ergothioneine biosynthesis

Sulfur-containing molecules are abundant in nature and in pharmaceutical and agrochemical industries. Many chemical and enzymatic strategies for C-S bond formation have been identified and used to create novel compounds. Recently, there has been a considerable amount of interest in direct C-H bond functionalization, especially in reactions which have been developed to directly transform C-H to C-S bonds. Such transformations have also been observed in nature, for example, in ergothioneine biosynthesis. In Chapter 1, a brief overview on sulfur-containing molecules and synthetic/biosynthetic strategies to synthesise these is given. Ergothioneine is a sulfur-containing derivative of histidine with antioxidant properties. In the following sections of this thesis, a novel biosynthetic enzyme for direct C-H to C-S bond transformation is described; the anaerobic ergothioneine biosynthetic enzyme, EanB. The enzymes for oxygen-dependent ergothioneine biosynthesis (EgtA-E) were described a few years ago. In Chapter 3, the identification of an oxygen-independent ergothioneine biosynthetic pathway is described. The pathway involves only two enzymes - the methyl transferase EanA and the sulfurtransferase EanB. In addition to the in vitro reconstitution of oxygen-independent ergothioneine biosynthesis, we could show that the extremely halophilic bacterium, Salinibacter ruber, an organism carrying genetic information only for anaerobic ergothioneine biosynthesis, could produce ergothioneine in similar concentrations as has been described for those carrying the genetic information for aerobic ergothioneine biosynthesis. This study was followed up by a structural and mechanistic investigation of the EanB-catalyzed C-S bond formation, described in Chapter 4. Based on structural and kinetic data, a mechanistic model for the direct C-H to C-S bond transformation catalyzed by EanB was elucidated. EanB is suggested to follow a ping-pong mechanism where, in a first step, a persulfide is formed on an active site cysteine residue of EanB and, in a second step, a sulfane sulfur is transferred to the unactivated carbon 2 of the imidazole ring of N-α-trimethylhistidine. The use of a single turnover assay allowed us to investigate the sulfur transfer from the enzyme to the substrate isolated from the formation of the active site persulfide. In Chapter 5, the focus is set on the non-heme iron-dependent ergothioneine biosynthetic enzyme, EgtB. EgtB catalyzes the oxygen-dependent C-S bond formation in aerobic ergothioneine biosynthesis. We designed substrate analogs to probe the binding interactions between the two substrates, N-α-trimethyl histidine and cysteine/γ-glutamyl cysteine. The result of this study led to the development of bisubstrates which revealed the importance of substrate alignment for efficient C-S bond formation. Additionally, we analyzed substrate binding and regulation of the SAM-dependent methyl transferase EgtD, the only enzyme common to all known ergothioneine biosynthetic pathways. This study, described in Chapter 6, provides a good basis for further inhibitor design. This thesis gives an insight into two different strategies of C-S bond formation in ergothioneine biosynthesis. Our findings contribute to a further understanding of the role of ergothioneine in vivo and its molecular mechanism of action

In Vitro Production of Ergothioneine Isotopologues

Ergothioneine is an emerging component of the redox homeostasis system in human cells and in microbial pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei. The synthesis of stable isotope-labeled ergothioneine derivatives may provide important tools for deciphering the distribution, function, and metabolism of this compound in vivo. We describe a general protocol for the production of ergothioneine isotopologues with programmable 2 H, 15 N, 13 C, 34 S, and 33 S isotope labeling patterns. This enzyme-based approach makes efficient use of commercial isotope reagents and is also directly applicable to the synthesis of radio-isotopologues

First evidence of ovothiol biosynthesis in marine diatoms

Ovothiols are histidine-derived thiols that are receiving a great interest for their biological activities in human model systems. Thanks to the position of the thiol group on the imidazole ring of histidine, these compounds exhibit unusual antioxidant properties. They have been revealing a very promising pharmacological potential due to their anti-proliferative and anti-inflammatory properties, as well as anti-fibrotic activities not always related to their antioxidant power. Ovothiols occur in three differentially methylated forms (A, B and C), isolated from ovary, eggs and biological fluids of many marine invertebrates, mollusks, microalgae, and pathogenic protozoa. These molecules are synthesized by two enzymes: the sulfoxide synthase OvoA and the sulfoxide lyase OvoB. OvoA catalyzes the insertion of the sulfur atom of cysteine on the imidazole ring of histidine, leading to the formation of a sulfoxide intermediate. This is then cleaved by OvoB, giving 5-thiohistidine, finally methylated on the imidazole ring thanks to the methyltransferase domain of OvoA. Recent studies have shown that OvoA homologs are encoded in a wide variety of genomes suggesting that ovothiol biosynthesis is much more widespread in nature than initially thought. Here we have investigated the OvoA occurrence in diatoms, one of the most abundant group of microalgae, dominating marine and freshwater environments. They are considered a very good model system for both biology/photophysiology studies and for biotechnological applications. We have performed comparative sequence and phylogenetic analyses of OvoA from diatoms, highlighting a high degree of conservation of the canonical domain architecture in the analyzed species, as well as a clear clustering of OvoA in the two different morphological groups, i.e. centric and pennate diatoms. The in silico analyses have also revealed that OvoA gene expression is modulated by growth conditions. More importantly, we have characterized the thiol fraction from cultures of the coastal centric diatom Skeletonema marinoi, providing the first evidence of ovothiol B biosynthesis in diatoms

Anaerobic Origin of Ergothioneine

Ergothioneine is a sulfur metabolite that occurs in microorganisms, fungi, plants, and animals. The physiological function of ergothioneine is not clear. In recent years broad scientific consensus has formed around the idea that cellular ergothioneine primarily protects against reactive oxygen species. Herein we provide evidence that this focus on oxygen chemistry may be too narrow. We describe two enzymes from the strictly anaerobic green sulfur bacterium Chlorobium limicola that mediate oxygen-independent biosynthesis of ergothioneine. This anoxic origin suggests that ergothioneine is also important for oxygen-independent life. Furthermore, one of the discovered ergothioneine biosynthetic enzymes provides the first example of a rhodanese-like enzyme that transfers sulfur to non-activated carbon

Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD

Ergothioneine is an emerging factor in cellular redox homeostasis in bacteria, fungi, plants, and animals. Reports that ergothioneine biosynthesis may be important for the pathogenicity of bacteria and fungi raise the question as to how this pathway is regulated and whether the corresponding enzymes may be therapeutic targets. The first step in ergothioneine biosynthesis is catalyzed by the methyltransferase EgtD that converts histidine into N-α-trimethylhistidine. This report examines the kinetic, thermodynamic and structural basis for substrate, product, and inhibitor binding by EgtD from <i>Mycobacterium smegmatis</i>. This study reveals an unprecedented substrate binding mechanism and a fine-tuned affinity landscape as determinants for product specificity and product inhibition. Both properties are evolved features that optimize the function of EgtD in the context of cellular ergothioneine production. On the basis of these findings, we developed a series of simple histidine derivatives that inhibit methyltransferase activity at low micromolar concentrations. Crystal structures of inhibited complexes validate this structure- and mechanism-based design strategy