%0 Journal Article %A María Victoria Revuelta %A Nicolas Stocchi %A Priscila Ailín Lanza Castronuovo %A Mariano Vera %A Arjen ten Have %T Substrate binding and specificity appear as major forces in the functional diversification of eqolisins %D 2017 %R 10.1101/167544 %J bioRxiv %P 167544 %X Background Eqolisins are rare acid proteases found in archaea, bacteria and fungi. Certain fungi secrete acids as part of their lifestyle and interestingly these also have many eqolisin paralogs, up to seven paralogs have been recorded. This suggest functional redundancy and diversification, which was the subject of the research we performed and describe here.Results We identified eqolisin homologs by means of iterative HMMER analysis of the NR database. The identified sequences were scrutinized for which we defined novel hallmarks, identified by molecular dynamics simulations of mutants of highly conserved positions, using the structure of an eqolisin that was crystallized in the presence of a transition state inhibitor. Four conserved glycines were shown to be required for functionality. A mutation of W67F is shown to be accompanied by the L105W mutation. Molecular dynamics shows that the W67 binds to the substrate via a π–π stacking and a salt bridge. The latter is stronger in the W67F/L105W double mutant. Additional likely fatal mutants are discussed.Upon sequence scrutiny we obtained a set of 233 sequences that in all likelihood lacks false positives. This was used to reconstruct a Bayesian phylogenetic tree. We identified 14 specificity determining positions. These occur in two pairs that are explained by structural constraints as well as two sub-networks of real specificity determining positions. The first sub-network is related to substrate specificity whereas the second sub-network seems to affect the dynamics of three loops that are involved in substrate binding. %U https://www.biorxiv.org/content/biorxiv/early/2017/08/04/167544.full.pdf