Functional Diversification of Tripeptidyl Peptidase and Endopeptidase Sedolisins in Fungi

Abstract

max)Sedolisins are acid proteases that are related with the basic subtilisins. They have been identified in all three superkingdoms but are not ubiquitous, although fungi that secrete acids as part of their lifestyle can have up to six paralogs. Both tripeptidylpeptidase (TPP) and endopeptidase activity have been identified and it has been suggested that these correspond to separate subfamilies.We studied eukaryotic sedolisins by computational analysis. A maximum likelihood tree shows three major clades of which two contain only fungal sequences. One fungal clade contains all known TPPs whereas the other contains the endosedolisins. We identified four cluster specific inserts (CSIs) in endosedolisins, of which CSIs 1, 3 and 4 appear as solvent exposed according to structure modeling. Part of CSI2 is exposed but a short stretch forms a novel and partially buried αhelix that induces a conformational change near the binding pocket. We also identified a total of 13 specificity determining positions (SDPs) that are divided over three SDP sub-networks. The major SDP network contains eight SDPs and modeling of virtual mutants suggests a key role for the W307A or F307A substitution. This is accompanied by a group of four SDPs that physically interact at the interface of the catalytic domain and the enzyme's prosegment. Modeling of virtual mutants suggests these SDPs are indeed required to compensate the conformational change induced by CSI2 and the A307. The additional small SDP networks appear to be linked to the major network all together explaining the hypothesizes functional diversification of fungal sedolisins.

Substrate binding and specificity appear as major forces in the functional diversification of eqolisins

“…However, since both sedolisins and eqolisins are typically active below pH 4, they 50 are interesting both from a fundamental and an applied angle. Here we study the eqolisins by means of molecular evolution and dynamics, a study on sedolisins we recently reported elsewhere [3].…”

“…Interestingly, these also show relatively few basic subtilisins. Recently we studied the sedolisins [3], here, we explore the eqolisin protein family in order to identify which residues are likely required for function and which could be involved in functional diversification. 95…”

“…This is also suggested by the fact that B. cinerea and S. sclerotiorum, as well as for instance many Aspergilli, secrete acids and seem to have relatively many eqolisins, as well as sedolisins and for 4 65 70 75 80 85 instance few basic subtilisins. Recently we studied the sedolisins [14], here, we explore the eqolisin protein family in order to identify which residues are likely required for function and which could be involved in functional diversification.…”