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.