Ex vivo metabolomics-A hypothesis-free approach to identify native substrate(s) and product(s) of orphan enzymes.

Abstract

Over the past decade, the number of fully sequenced genomes has increased at an awe-inspiring pace. Similarly, the quality and scope of tools for the prediction of both protein structure and function has seen vast improvements. However, to pinpoint the exact function of a protein, for instance the exact reaction catalyzed by an enzyme, experimental evidence is crucial. At the same time, this step is the main bottleneck when generating a conclusive model for the function of an enzyme and to interpret its function in a physiological context. Hence, a comprehensive experimental strategy for functional annotation of enzymes that is as efficient as possible is required. Ex vivo metabolomics is a powerful non-targeted approach that overcomes several of the challenges inherent to in vitro characterization of enzymes with unknown functions. By incubating the recombinant enzyme of interest in a quasi-native metabolite extract from its tissue of origin under specific environmental and developmental conditions, the complete native substrate range can be tested in a single assay. This unlocks compounds that are commercially unavailable or otherwise difficult to procure. Coupled with non-targeted metabolomics analysis, ex vivo has the capability to test for and identify even unexpected substrates and assign the respective products of the enzymatic reaction.