Evolution and Structure-Function Prediction of Eqolisins in Fungi

STOCCHI, N; REVUELTA MV; VERA M; ARJEN TEN HAVE

Bahía Blanca

Congreso; 6to Congreso Argentino de Bioinformática y Biología Computacional; 2015

Asociacion Argentina de Bioinformática y Biología Computacional

p { margin-bottom: 0.1in; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; text-align: left; }p.western { font-family: "Liberation Serif","Times New Roman",serif; font-size: 12pt; }p.cjk { font-family: "Droid Sans Fallback"; font-size: 12pt; }p.ctl { font-family: "FreeSans"; font-size: 12pt; }a:link { }BackgroundBotrytiscinereais a phytopathogenic fungus that acidifies its environment (pH<4),which has been shown to be related with the spectrum of secretedhydrolytic enzymes that are involved in the degradation of the host´scell wall. Among these are about 20 proteases that belong to 3protein families that are subject to functional redundancy anddiversification. The eqolisins or glutamic peptidasas (G1) form anovel family of acid endopeptidases and are encoded by four genes inB.cinerea.Point mutation analyses revealed its catalytic site is formed by acatalytic dyad (Q53, E136, hence the name eqolisin), the catalyticmechanism is known, and structures, including a structure with atransition state inhibitor, are available. Eqolisins are composed oftwo seven-stranded anti-parallel β-sheets which form a convexβ-sandwich structure. It has two loops, the ?beta loop? and the ?70´s loop? whichare likely involved in substrate interaction. Here we report a studyon the evolution, and more particularly, the functional diversifationof eqolisins in fungi.ResultsEqolisinswere found in a few archaea, a few bacteria and a few fungi,surprisingly they were not found in non-fungal eukaryotes. Phylogenyclearly separates the eukaryotic and prokaryotic eqolisins and it istherefore likely that fungi have obtained eqolisins from bacteria byhorizontal gene transfer. Given the novelty of the enzyme and thevery restricted taxonomical distribution, only few sequences wereidentified. In order to obtain a dataset lacking false positives, wefirst performed a structure-function analysis directed at removingdoubtfull sequences. In addition to the catalytic residues, weidentified several highly conserved sites such as W67, involved inbinding substrate, and a number of glycines present in the innerbeta-sheet, that are likely involved in the dynamics of the enzyme. Usinga strict dataset and SDPox we identified 20 cluster deteminingpositions. A comparison with data regarding mutual information,determined by Mistic, revealed two pairs of positions important instructure, as well as two small networks of connected specificitydetermining positions. Structural analysis suggests one of these isinvolved in substrate specificity, identifying a group that requiresan acidic amino acid at the S3´ position, and a group that likely haslower substrate specificity. The other small network corresponds witha disulphide-bridge anchored loop that appears to cooperate with the70s loop in enzyme dynamics during substrate binding. Moleculardynamics studies are underway in order to shed more light on how thenovel identified loop interact with the 70s loop during substrateaccomodation.ConclusionDespitethat eqolisins are rare enzymes, they have undergone at least twoprocesses of functional diversification, one directed by substratespecificity and one that appears to affect enzyme dynamics.