How structure guides function: ergothioneine anabolism and catabolism formylglycine generating enzyme methyl transferases

Abstract. A detailed understanding of the underlying mechanisms determining the chemistry of all involved enzymes is crucial to understand any biological system and eventually gives the opportunity to manipulate such systems. Macromolecular X-ray crystallography is still an invaluable technique in the biophysical toolbox of a biochemist to relate macroscopic observations, such as activity or stability back to the molecular composition of an enzyme on atomic level. Awareness of the molecular structure of biological catalysts and an in-detail understanding of the individual contribution of particular amino acids to enzyme catalyzed chemical transformations is of crucial importance in life sciences. Being able to identify and understand structure – function relationships within an enzyme is a key prerequisite on biochemists ambition to decipher the underlaying mechanistic details of the catalyzed reaction. Structural insights inform for mutational studies and allow to extract motifs of key residues from primary sequence to inform bioinformatic studies. Even subtle differences in the overall three- dimensional fold of a protein or conformational changes at an individual amino acid residue level enable biochemists to predict functional changes or provide explanations for observed phenomena.
This thesis adresses questions about the structural details of enzymatic systems at two different levels. Fristly, crystal structures of enzymes, that are part of biosynthetic pathways, are discussed (a) to derive a sound mechanistic model, (b) to predict and explain substrate specificity and (c) to leverage amino acid sequence motifs for bioinformatic approaches to reveal relations among enzyme classes or probe the evolutionary context.
Secondly, X-ray crystallography was applied in a more sophisticated approach to decipher specific intermediates of the catalytic cycle of the formylglycine generating enzyme (FGE). FGE is a copper dependent oxidase, which catalyzes the thiol to thione oxidation of a peptidyl cysteine substrate. The three-dimensional fold of FGE, its mode of copper co-factor coordination in a thiol rich environment and its strategy to de-couple oxygen binding and activation are fascinatingly uncommon among copper dependent oxidases. In-depth characterization of specific states of the catalytic cycle allows us to formulate a sound mechanistic proposal attribute particular functions to some unusual FGE-fold elements.
Applying the first strategy, we for example characterized EanB, an enzyme that catalyzes the central C–S bond forming step in anaerobic ergothioneine synthesis. EanB catalyzes this C– S bond formation via a previously unknown oxidative sulfurization mechanism. Based on sequence space analysis we suggest that this mechanism may be widespread among rhodanese-like enzymes. The anaerobic ergothioneine biosynthesis pathway complements intensive investigations into the biosynthesis of this small sulfur containing metabolite over the last decade, which revealed at least three different pathways of oxygen dependent ergothioneine biosynthesis.
In contrast to the intensive investigation of the enzymes involved in the biosynthesis and uptake of ergothioneine less is known about catabolic pathways of ergothioneine. We thus structurally and biochemically characterized a trimethyl ammonia lyases (ergothionase) which is the first enzyme in the catabolic pathway of ergothioneine. Ergothionases catalyzes the 1,2- elimination of trimethyl amine from ergothioneine. In contrast to phylogenetically related MIO- dependent aromatic ammonia lyases, which catalyze the common first step in histidine catabolism, ergothionases follow a sufficiently different mechanism. We identified substrate specificity determining sequence motifs, which allowed to start classifying the trimethyl ammonia lyase enzyme family. Within that family we identified discrete substrate specificities as well as independent evolutionary emergences. Trimethyl ammonia lyases of different phylogenetic origin show consistently similar substrate specificitys and a preserved leaving group activation mechanism.
Starting off with the elimination of the trimethyl amine group the catabolism of ergothioneine all the way to L-glutamate was identified to be encoded in a five-enzyme pathway. The second and third enzyme of the ergothioneine catabolic pathway, a thiourocanate hydratase and a thiohydantoin desulfurase, were structurally and functionally characterized. No homolog of thiohydantoin desulfurase had been structurally described before.
Furthermore, structural and function characterization of a SAM-dependent halide methyl transferase, that plays a key role in the biosynthesis of methyl halides, revealed, that subtle changes of the active site composition severely affects substrate specificity. Structural insights are valuable since this class of enzyme has high application potential in biocatalytic processes.