Phylogenetic Analysis
The inversion of the “active site” configuration between HSA and BSA
mentioned before is evidence of the evolutionary conservation of
acid-basic properties required to sustain the catalytic condensation of
the aldol. How many times did this inversion occur in the evolutionary
history of the seroalbumin studied? To answer this question we used the
phylogenetic tree in Figure 2 and applied ancestral reconstruction
techniques to infer ancestral states for the equivalent positions to Lys
199 and Arg 222 from HSA (represented as “KR”). As it is derived from
Figure 2, we show in given internal nodes the estimated ancestral states
for those positions. For example, the ancestral node involving all
mammals contains two Lys in equivalent positions to Lys 199 and Arg 222
(shown as “KK” in the tree of Figure 2). Among the organisms in our
phylogenetic tree, we can find different combinations like “KR”
(Homo sapiens, Sus scrofa, and Rattus norvegicus ), “RK”
(Bos bovis and Capra hircus ), or “RR” (Chinchilla
Lanigera, Oryctolagus cuniculus, and Bubalus bubalis ). The occurrence
of these shifts of amino acid with the conservation of the corresponding
pKa shifts possibly indicates the biological importance of these
residues.
Finally, for a deeper analysis, we estimated the random occurrence of
these shifts using the phylogenetic tree in Figure 2 and setting the
ancestral state of these two positions as “KK” as derived from the
ancestral reconstruction results. Then we simulated the evolutionary
history of sequences using Seq-gen32and counted the occurrence at the tips of the different combinations.
Comparing the frequencies in the natural sequences and in those
simulated with Seq-gen we obtained that the occurrence for KR and RK
pairs are significantly greater (p-value < 0.001) in the
observed sequences than in the simulated sequences. Our result suggests
that this inversion is structurally constrained indicating a putative
functional adaptation.