Assays developments for the determination of the enzymatic activity of aldehydes dehydrogenases of invertebrate chordates.

This project represents an extension of the paradigms that we created from our molecular simulation studies to understand the frequent structure and function changes of aldehyde dehydrogenases (ALDH) during evolution. The ALDHs form a superfamily of proteins that catalyze the oxidation of several aldehydes, but the evolutionary origins of their substrate preference are unknown. Despite having a high sequence identity, two of these ALDHs, the ADLH1, and ALDH2, exhibit distinct functional roles of cellular signaling and detoxication, respectively. Through previous computational and phylogenetic analysis, we found that, interestingly, the ALDH1s of invertebrate organisms (Branchiostoma oridaeand Ciona intestinalis) show structural features more similar to their ALDH2s than the typical ALDH1. It suggests that these divergent ALDH1sevolved to provide small aldehydes detoxication pattern, what seems to represent the ancestral eukaryotic ALDH2s function. Our analysis also identied three aminoacidsignatures, located internally in the substrate entry channel (SEC), that distinguishes the ALDH1 from ALDH2. Thus, we nd that ALDH1s have a wide, open and unobstructed SEC, consistent with the fact that these enzymes catalyze bulky long-chainaldehydes, like retinaldehyde, a precursor of important signaling pathways. In contrast, the ALDH2s have a small and constricted SEC, consistent with the degradation function of small aldehydes, like the toxic metabolite acetaldehyde. In this project, our objective is to understand the functional and structural correlation between ALDH1 and ALDH2 found in B. oridaeand C. intestinalisto discover and comprehend their functional and evolutionary roles in chordates. Specically, we will test the hypothesis that the three signatures described above are the fundamental core of the substrate preference, based on the signature origin. If conrmed experimentally, this will be a pioneeringexample of evolutionary molecular reversion, impacting directly on the current interpretations of the controversial Dollos Law of Irreversibility. (AU)