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.