Introduction
First hypotheses on the origin of protein promiscuous activities proposed them as the starting point for new protein activities1. Many directed evolution studies showed that protein plasticity2, a feature of many promiscuous proteins and complexes3, is an important factor for random mutations to endure selection throughout generations. In the same sense, protein backbone rearrangements could be responsible for changes in the protein’s functionality4. These observations may be expected taking into consideration the relationship between conformational diversity, biological function and evolvability5.
Tawfik and co-workers defined promiscuity as the capability of a protein to catalyze reactions different from those it has evolved to sustain6. From this evolutionary perspective many cases of promiscuous behavior in proteins, and their functionality in general, could be rethought. The serum albumins constitute one of these very interesting cases of catalytic promiscuity that need to be revisited. Members of this family show a large structural diversity in spite of global sequence conservation7. Moreover, they differ in the ability to perform complex promiscuous activities, their catalytic ability has been tested for several reactions8–12and they have also been used as biocatalysts in organic reactions such as the Henry reaction, the Kemp elimination and the cross aldol condensation, to give a few examples10,13,14.
Human Serum Albumin (HSA) is the main protein in plasma, binds multiple ligands15, and has recently emerged as a very important drug carrier16,17. This single-chain protein has several high-affinity binding sites, however the majority of the drugs and ligands bind to the so named Sites I and II18. In particular, residues Lys 199, Arg 410, Tyr 411, Cys 34, and Lys 195 from HSA are described as some of the important ones, not only for ligand binding but also for catalysis19–21. Among the best characterized catalytic activities described are the esterase-like activities and the thioesterase activity, shared with other albumins as Bovine Serum Albumin (BSA)9. Such multiple catalytic capacities can be associated with some structural cavity features such as the presence of activated amino acids (with abnormal pKas) in mostly hydrophobic environments, which would increase the catalytic repertoire favoring the attack of substrates and allow creating the microenvironment suitable for catalysis22,23. The structural determinants that allow such a diversity of reactions to be carried out in the same active site of albumins, with minimal variations between species, are still as poorly understood as the biological implications and evolutionary origin of such activities. If promiscuous activities arise spontaneously and due to structural or mechanistic similarity, there should be no evolutionary imprint that demonstrates functional adaptation2,24.
Given this and due to the numerous promiscuous reactions described for albumin, we wonder if these activities occur by chance or if they actually have physiological or adaptive bases that remain as yet uncharacterized. Is albumin, therefore, an enzyme of activity not yet characterized? In the present work, we analyze structural and evolutionary patterns of HSA, BSA, RabSA (Rabbit Serum Albumin), PSA (Pig Serum Albumin), and RSA (Rat Serum Albumin) as representatives of the albumin family to bring some light to these questions. For these purposes, we used evolutionary and structural analysis to characterize the probable presence of functional adaptations during evolution in positions previously reported as supporting the promiscuous behavior in albumins. To evaluate this hypothesis we used the cross aldol condensation of acetone and p -formylbenzonitrile, previously assayed with BSA14, now extended to other members of the albumin family.