ochroleuca, as well as 2 additional proteins from M. brunnea and A. montagnei. While phylogenetic GW3965 manufacturer reconstruction by maximum likelihood indicated strong support for a monophyletic clade formed by the cluster members (Figure 4), positioning of the resulting

clade within a/b-hydrolase phylogeny was poorly supported and thus remains uncertain. QNZ datasheet Figure 4 Maximum likelihood phylogenetic tree of zearalenone lactonohydrolase homologs from divergent filamentous fungi. Bootstrap support is indicated below bifurcations (1000 bootstrap iterations). Tree was based on 245 distinct patterns within a trimmed alignment of full length protein sequences (see: Methods section). Homology modelling and comparative structure analysis The created homology models uncovered similarities in the active site pocket, as detected by fpocket[15]. In all of the modelled structures, the active site pocket is strongly hydrophobic under normal conditions – likely the catalysis is enabled by allowing access to the active

site (conformational changes involving cap domain) which allows the reaction to proceed by standard mechanism involving forming a transient oxyanion hole and subsequent cleavage of the lactone ring (Figure 5). While homology-based models are likely insufficient for elucidation of full sequence of events during substrate binding and catalysis (both the variable cap domain e.g. [16, 17] and surrounding loops [18] are involved in controlling and fine-tuning substrate access), we were nevertheless able to ascertain the key functional residues involved. Figure 5 Superposed structures of template 2XUA (3-oxoadipate PF-3084014 chemical structure lactonase; catalytic domain colored in green, cap domain colored in yellow) and homology models for zearalenone

lactonohydrolase homologs from multiple species (see corresponding alignment on Figure 6 ). Coloring is based on RMSD between superposed Ca atoms (blue – best, red – worst; gray parts not included in superposition). Our identification of the catalytic triad conflicts with the initial proposition of Takahashi-Ando [11] that active site is formed by S102-H242-D223 (numeration by alignment in Figure 6). Typically, the nucleophilic attack of hydrolase enzyme Inositol monophosphatase 1 is facilitated by interaction of histidine with acidic residue (third member of catalytic triad). This role, according to all our homology-based models cannot be fulfilled by D223 (residue located distantly to active site – Figure 7). Figure 6 Multiple alignment of protein sequences corresponding to: template structure 2XUA (3-oxoadipate lactonase), template structure 2Y6U (peroxisomal epoxide hydrolase Lpx1) and lactonase homologs from examined isolates (AN154, AN169, AN171), as well as reference sequences from Bionectria ochroleuca (GBK:AB076037), Apiospora montagnei (JGI:58672) and Marsonnina brunnea (MBM_00923 = GBK:EKD21810).