Ised the conserved amino acid differences of surface exposed residues within each of the protein families. No conspicuous regions of conserved differences were detected which would indicate sites of interaction with different protein partners.The active site cavityThe active site is present in a largely hydrophobic pocket of ?volume 336 A3, with a classical serine hydrolase catalytic triad at its base (composed of Ser95-His246-Asp217). The KAI2 catalytic ?pocket is smaller than the catalytic pocket of DAD2 (448 A3), but it is still sufficiently large to accommodate the synthetic strigolactone GR24. The two pockets are highly similar in terms of overall shape and amino acid composition (Figures 3A and 3B). No obvious active site cavity features account for a difference between KAIThe Structure of KAIFigure 4. The KAI2 catalytic triad. A. The catalytic residues Ser95His246-Asp217 are hydrogen bonded in a classical arrangement for a serine hydrolase (Residues are shown in stick representation, coloured by atom type). B. CH…O hydrogen bonding between histidine Ce1 and carbonyl oxygens. KAI2 (blue), DAD2 (purple) and RsbQ (brown) all superimposed using the imidazole ring of the catalytic histidine of each respective protein. KAI2, unlike DAD2 and RsbQ, has unfavourable hydrogen bonding geometry between the catalytic His Ce1 and the carbonyl oxygen. This hydrogen bond is important for serine hydrolase activity [37,38]. doi:10.1371/journal.pone.0054758.gFigure 5. The second pocket of KAI2. A. The two pockets of KAI2a are separated internally by the aromatic side-chain of Phe26. The active site residues can be seen in the primary pocket (left). Both pockets are solvent accessible. B. The active site cavity of DAD2 and the adjacent small, non-solvent accessible pocket. doi:10.1371/journal.pone.0054758.gand D14 in terms of karrikin binding. Six of the seven cavity-lining phenylalanine residues are conserved between the two get Oltipraz proteins (Figures 3A and 3B). The non-conserved 166518-60-1 residue within these is Tyr124, which replaces Phe125. The hydroxyl group of this side ?chain occludes a small pocket (41 A3) proximal to the catalytic residues. In DAD2, this small pocket is connected to the main cavity, helping to explain the discrepancy in size between the KAI2 and DAD2 main cavity volumes. This specific Tyr/Phe substitution is conserved within KAI2 and D14 protein families respectively (Figure 3C) suggesting that the two proteins may differ in their natural substrates/ligands.Catalytic residuesThe catalytic triad of KAI2 is observed with either a Tris buffer molecule (KAI2a) or glycerol molecule (KAI2b) nearby. As the structure of a PMSF-adduct of RsbQ had previously been reported [25], we attempted extensive incubation of KAI2 with PMSF, but were unable to detect any covalent modification at Ser95 either in crystals or by electrospray mass-spectrometry. This observation is not unprecedented for catalytically active serine hydrolases [35,36]. In an attempt to rationalise the inactivity of KAI2 towards PMSF, we scrutinised the conformation of the catalytic residues. The Ser-His-Asp triad residues are hydrogen bonded in a classical conformation for active hydrolysis (Figure 4A)[34]. However, the imidazole side-chain of His246 is in a different plane to that observed in DAD2 and RsbQ, coordinating to Od1 of Asp217 as opposed to Od2. Furthermore, a discrepancy in the coordination of Ce1 of His246 in KAI2 compared to that of active serine hydrolases, including DA.Ised the conserved amino acid differences of surface exposed residues within each of the protein families. No conspicuous regions of conserved differences were detected which would indicate sites of interaction with different protein partners.The active site cavityThe active site is present in a largely hydrophobic pocket of ?volume 336 A3, with a classical serine hydrolase catalytic triad at its base (composed of Ser95-His246-Asp217). The KAI2 catalytic ?pocket is smaller than the catalytic pocket of DAD2 (448 A3), but it is still sufficiently large to accommodate the synthetic strigolactone GR24. The two pockets are highly similar in terms of overall shape and amino acid composition (Figures 3A and 3B). No obvious active site cavity features account for a difference between KAIThe Structure of KAIFigure 4. The KAI2 catalytic triad. A. The catalytic residues Ser95His246-Asp217 are hydrogen bonded in a classical arrangement for a serine hydrolase (Residues are shown in stick representation, coloured by atom type). B. CH…O hydrogen bonding between histidine Ce1 and carbonyl oxygens. KAI2 (blue), DAD2 (purple) and RsbQ (brown) all superimposed using the imidazole ring of the catalytic histidine of each respective protein. KAI2, unlike DAD2 and RsbQ, has unfavourable hydrogen bonding geometry between the catalytic His Ce1 and the carbonyl oxygen. This hydrogen bond is important for serine hydrolase activity [37,38]. doi:10.1371/journal.pone.0054758.gFigure 5. The second pocket of KAI2. A. The two pockets of KAI2a are separated internally by the aromatic side-chain of Phe26. The active site residues can be seen in the primary pocket (left). Both pockets are solvent accessible. B. The active site cavity of DAD2 and the adjacent small, non-solvent accessible pocket. doi:10.1371/journal.pone.0054758.gand D14 in terms of karrikin binding. Six of the seven cavity-lining phenylalanine residues are conserved between the two proteins (Figures 3A and 3B). The non-conserved residue within these is Tyr124, which replaces Phe125. The hydroxyl group of this side ?chain occludes a small pocket (41 A3) proximal to the catalytic residues. In DAD2, this small pocket is connected to the main cavity, helping to explain the discrepancy in size between the KAI2 and DAD2 main cavity volumes. This specific Tyr/Phe substitution is conserved within KAI2 and D14 protein families respectively (Figure 3C) suggesting that the two proteins may differ in their natural substrates/ligands.Catalytic residuesThe catalytic triad of KAI2 is observed with either a Tris buffer molecule (KAI2a) or glycerol molecule (KAI2b) nearby. As the structure of a PMSF-adduct of RsbQ had previously been reported [25], we attempted extensive incubation of KAI2 with PMSF, but were unable to detect any covalent modification at Ser95 either in crystals or by electrospray mass-spectrometry. This observation is not unprecedented for catalytically active serine hydrolases [35,36]. In an attempt to rationalise the inactivity of KAI2 towards PMSF, we scrutinised the conformation of the catalytic residues. The Ser-His-Asp triad residues are hydrogen bonded in a classical conformation for active hydrolysis (Figure 4A)[34]. However, the imidazole side-chain of His246 is in a different plane to that observed in DAD2 and RsbQ, coordinating to Od1 of Asp217 as opposed to Od2. Furthermore, a discrepancy in the coordination of Ce1 of His246 in KAI2 compared to that of active serine hydrolases, including DA.
