The enzymatic landscape of ergothioneine degradation

An understanding of the molecular basis of the biotransformation of natural metabolites is
necessary to dissect their physiological functions. Ergothioneine, a widely distributed derivative
of histidine, is involved in numerous biological processes in microorganisms and animals,
however, the molecular mechanisms of these processes remain obscure. This work provides
insights into the bacterial utilization of ergothioneine in order to shed light on the biochemical
properties of this molecule.
Bacteria degrade ergothioneine via a five-step catabolic pathway. This pathway is essentially
adapted from two catabolic pathways of primary metabolism, however, has evolved some
exceptional enzymatic features and activities. Thus, the first enzyme in ergothioneine degradation
– ergothionase – is the first enzyme that has been identified to catalyze trimethylamine
elimination and opens a scope of cofactor-independent C-N bond-cleaving enzymes among the
homologous aromatic amino-acid lyases. The second enzyme, thiourocanate hydratase, is the
second known enzyme to utilize a common redox cofactor NAD+ in a very unusual way,
exposing the electrophilic properties of this cofactor. Subsequently, the third step of non-redox
desulfurization is catalyzed by the enzyme, thiohydantoin desulfurase, which is the very first
enzyme of its class with such activity. Each of these enzymes represents a novel function and
discloses the diversity of enzymatic families they belong to.
In this thesis, we provide a detailed characterization of the enzymes involved in ergothioneine
catabolism by dissection their catalytic mechanisms and crystal structures. This allows to
identify homologous enzymes and pathways in various species with high precision and
estimate ergothioneine distribution in nature. Moreover, we discovered and characterized two
bacterial transporting systems that are involved in the high-affinity uptake of ergothioneine from
the environment. This research provides an excellent platform to examine the impact of bacterial
activity on the environmental availability of ergothioneine. Inspired by the unique mechanistic
features of these enzymes, we delve into the diversity of the reactivities found in homologous
enzymes. This approach led to the discovery of the degradation pathways of other histidine
derivatives, such as Nτ- and Nα-monomethylhistidines.
Furthermore, we discovered a completely new ergothioneine biosynthetic pathway, utilized by
bacteria living in the abyssal zone. This pathway occurs mainly in anaerobic organisms living
in conditions that reflect the primitive Earth and is yet another indication of ergothioneine
prevalence in Nature and its importance to many life forms.
Finally, we developed an enzymatic approach for the synthesis of ergothioneine isotopologues.
The ease of implementation of the procedure allows easy access to isotopically labeled
ergothioneine as a powerful tool to study ergothioneine functions both in vivo and in vitro.