Decipher enzymes from human microbiota for drug discovery and development.

The human microbiota plays an important role in human health and contributes to the metabolism of therapeutic drugs affecting their potency. However, the current knowledge on human gut bacterial metabolism is limited and lacks an understanding of the underlying mechanisms of observed drug biotransformations. Despite the complexity of the gut microbial community, genomic and metagenomic sequencing provides insights into the diversity of chemical reactions that can be carried out by the microbiota and poses new challenges to functionally annotate thousands of bacterial enzymes. Here, we outline methods to systematically address the structural and functional space of the human microbiome, highlighting a combination of in silico and in vitro approaches. Systematic knowledge about microbial enzymes could eventually be applied for personalized therapy, the development of prodrugs and modulators of unwanted bacterial activity, and the further discovery of new antibiotics.

Discovery and Characterization of the Metallopterin-Dependent Ergothioneine Synthase from Caldithrix abyssi.

Ergothioneine is a histidine derivative with a 2-mercaptoimidazole side chain and a trimethylated α-amino group. Although the physiological function of this natural product is not yet understood, the facts that many bacteria, some archaea, and most fungi produce ergothioneine and that plants and animals have specific mechanisms to absorb and distribute ergothioneine in specific tissues suggest a fundamental role in cellular life. The observation that ergothioneine biosynthesis has emerged multiple times in molecular evolution points to the same conclusion. Aerobic bacteria and fungi attach sulfur to the imidazole ring of trimethylhistidine via an O2-dependent reaction that is catalyzed by a mononuclear non-heme iron enzyme. Green sulfur bacteria and archaea use a rhodanese-like sulfur transferase to attach sulfur via oxidative polar substitution. In this report, we describe a third unrelated class of enzymes that catalyze sulfur transfer in ergothioneine production. The metallopterin-dependent ergothioneine synthase from Caldithrix abyssi contains an N-terminal module that is related to the tungsten-dependent acetylene hydratase and a C-terminal domain that is a functional cysteine desulfurase. The two modules cooperate to transfer sulfur from cysteine onto trimethylhistidine. Inactivation of the C-terminal desulfurase blocks ergothioneine production but maintains the ability of the metallopterin to exchange sulfur between ergothioneine and trimethylhistidine. Homologous bifunctional enzymes are encoded exclusively in anaerobic bacterial and archaeal species.

Bacterial Degradation of Nτ-Methylhistidine.

The first three enzymatic steps by which organisms degrade histidine are universally conserved. A histidine ammonia-lyase (EC 4.3.1.3) catalyzes 1,2-elimination of the α-amino group from l-histidine; a urocanate hydratase (EC 4.2.1.49) converts urocanate to 4-imidazolone-5-propionate, and this intermediate is hydrolyzed to N-formimino-l-glutamate by an imidazolonepropionase (EC 3.5.2.7). Surprisingly, despite broad distribution in many species from all kingdoms of life, this pathway has rarely served as a template for the evolution of other metabolic processes. The only other known pathway with a similar logic is that of ergothioneine degradation. In this report, we describe a new addition to this exclusive collection. We show that the firmicute Bacillus terra and other soil-dwelling bacteria contain enzymes for the degradation of Nτ-methylhistidine to l-glutamate and N-methylformamide. Our results indicate that in some environments, Nτ-methylhistidine can accumulate to concentrations that make its efficient degradation a competitive skill. In addition, this process describes the first biogenic source of N-methylformamide.

In Vitro Reconstitution of a Five-Step Pathway for Bacterial Ergothioneine Catabolism.

Ergothioneine is a histidine-derived sulfur metabolite that is biosynthesized by bacteria and fungi. Plants and animals absorb ergothioneine as a micronutrient from their environment or nutrition. Several different mechanisms of microbial ergothioneine production have been described in the past ten years. Much less is known about the genetic and structural basis for ergothioneine catabolism. In this report, we describe the in vitro reconstitution of a five-step pathway that degrades ergothioneine to l-glutamate, trimethylamine, hydrogen sulfide, carbon dioxide, and ammonia. The first two steps are catalyzed by the two enzymes ergothionase and thiourocanate hydratase. These enzymes are closely related to the first two enzymes in histidine catabolism. However, the crystal structure of thiourocanate hydratase from the firmicute Paenibacillus sp. reveals specific structural features that strictly differentiate the activity of this enzyme from that of urocanate hydratases. The final two steps are catalyzed by metal-dependent hydrolases that share most homology with the last two enzymes in uracil catabolism. The early and late part of this pathway are connected by an entirely new enzyme type that catalyzes desulfurization of a thiohydantoin intermediate. Homologous enzymes are encoded in many soil-dwelling firmicutes and proteobacteria, suggesting that bacterial activity may have a significant impact on the environmental availability of ergothioneine.

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.

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 Mycobacterium smegmatis. 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.