Nt was not performed at an PKCδ Activator drug optimal pH for the enzymatic reaction, or that the utilised substrate had a low binding affinity for the enzyme, thus making it energetically unfavourable to fit into a plausible active site. We need to note that Cip1 was characterised with all the similar substrate and at the very same pH optimum because the identified H. jecorina glucoronan lyase. Determination of Cip1 lyase activity might be a matter of finding the appropriate substrate and/or adjusting the pH.Functions and comparative evaluation of Cip1 to other protein structuresA structure similarity search with all the structure coordinates of Cip1 against all known and public protein structures revealed a higher degree of structural similarity involving Cip1 and also the protein structures of CsGL, a glucuronan lyase from H. jecorina (PDB ID: 2ZZJ), [12] and vAL-1, an alginate lyase in the Chlorella virus (PDB ID: 3A0N) [13]. The root-mean-square deviation (RMSD) values for these structures when superposed with the Cip1 ?structure, utilising the program Lsqman [14], were 1.54 A (for ?158 matched Ca atoms) and 1.98 A (for 143 matched Ca atoms), respectively. Some similarity was also located with the structure ofCrystal Structure of Cip1 from H. jecorinaFigure eight. Cip1 pocket that binds ethylene glycol. With Arg100 (lime green) forming certainly one of the walls, Thr85, Glu194, His83 and Tyr196 together produce the rest of a modest pocket on one side in the plausible active site cleft, in which an ethylene glycol (dark green) is identified inside the structure of Cip1. To facilitate comparison of figures, Gln104 is also shown (lime green). Electron density is contoured at a amount of 1.0 sigma ?(0.4 electrons/A3). doi:ten.1371/journal.pone.0070562.gCsCBM27-1, a protein having a CBM of family 27 from Caldicellulosiruptor saccharolyticus (PDB ID: 1PMH) in complex with a mannohexaose molecule [10]. Two regions stand out when comparing Cip1 to these three structures, namely the two regions described above as the “grip” motif and also the plausible active internet site cleft. Cip1 has two prospective substrate binding residues in typical using the Chlorella alginate lyase within the possible substrate-binding cleft. One is Gln104, corresponding to Gln120 inside the alginate lyase. This residue interacts with bound D-glucuronic acid within the structure on the Chlorella alginate lyase at pH 7 (PDB ID: 3A0N) (Figure 7a). The H. jecorina glucuronan lyase also has a glutamine at this position but no substrate was modelled into the structure. The other possible substrate-binding residue is definitely an arginine at position one hundred in Cip1, corresponding to Arg116 in the alginate lyase. This residue is located at the bottom of the active website cleft inside the Chlorella alginate lyase and interacts with the bound substrate at pH 10 (PDBID: 3IM0) (Figure 7). Rather of an arginine, the H. jecorina glucuronan lyase has a methionine at this position. Two Cip1 residues, Asp116 and His98, are positioned in the vicinity of the active site glutamine and arginine and both are modelled with dual conformations, which indicate that the region is dynamic (Figure 7). Gln104, Arg100, His98 and Asp116 are marked in orange in the sequence alignment in Figure 1. Even though the two lyase structures described above show a lot of charged residues lining the α4β7 Antagonist Biological Activity potential active site cleft, using the most hydrophobic ones being tyrosines, CsCBM27-1 is dependent upon three tryptophan residues to bind its mannohexaose substrate [10]. Since the residues lining the plausible active internet site cleft in Cip1 are mostly charge.
