Journal:Acta Cryst D:S2059798319000676
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<scene name='80/806393/Cv/20'>Active-site of PCPdr with pG binding residues shown in ball-and-sticks and bound pG is shown in magenta ball-and-sticks</scene>. Water molecules are shown as red spheres. The presence of pG in the active site identifies the structural features that enable this enzyme to be a pG specific peptidase. pG is bound into a pocket formed by both hydrophobic and polar residues, Phe9, Phe12, Asn18, Val45, Gly70, Leu71, Tyr142, Val143, Cys144 and His169. The <scene name='80/806393/Cv/12'>residues of loop-A (residues 9-19)</scene> play a particularly important role in the pG binding. The <scene name='80/806393/Cv/13'>phenyl groups of Phe9 and Phe12 form stacking interactions with the aliphatic 2-pyrrolidone moiety of pG</scene> while the <scene name='80/806393/Cv/14'>ND2 of Asn18 forms a hydrogen bond with 2' oxygen of pyrrolidone moiety of pG</scene>. These residues are conserved in all the known PCPs I, including those from mammals. The residues from other loops also contribute significantly towards pG binding. The <scene name='80/806393/Cv/15'>main chain nitrogen and oxygen of Leu71 form two hydrogen bonds with OE and NH of pG</scene>, respectively. The adjacent residue, <scene name='80/806393/Cv/16'>Gly70</scene>, forms van der Waals contacts with OE of pG. Other van der Waals contacts for the pG binding are formed by the side chains of <scene name='80/806393/Cv/17'>Tyr142, Val143, Cys144 and Val45</scene>. The 5' carboxylate group of pG is held by several ionic interactions: <scene name='80/806393/Cv/18'>one of its carboxylate oxygen forms a hydrogen bond with NE2 of His169</scene> while the <scene name='80/806393/Cv/19'>other carboxylate oxygen interacts with the main chain N of catalytic Cys144 and a water molecule</scene>. This water molecule is further stabilized by its interactions with the residues Tyr142, Asn145, and Ala139. | <scene name='80/806393/Cv/20'>Active-site of PCPdr with pG binding residues shown in ball-and-sticks and bound pG is shown in magenta ball-and-sticks</scene>. Water molecules are shown as red spheres. The presence of pG in the active site identifies the structural features that enable this enzyme to be a pG specific peptidase. pG is bound into a pocket formed by both hydrophobic and polar residues, Phe9, Phe12, Asn18, Val45, Gly70, Leu71, Tyr142, Val143, Cys144 and His169. The <scene name='80/806393/Cv/12'>residues of loop-A (residues 9-19)</scene> play a particularly important role in the pG binding. The <scene name='80/806393/Cv/13'>phenyl groups of Phe9 and Phe12 form stacking interactions with the aliphatic 2-pyrrolidone moiety of pG</scene> while the <scene name='80/806393/Cv/14'>ND2 of Asn18 forms a hydrogen bond with 2' oxygen of pyrrolidone moiety of pG</scene>. These residues are conserved in all the known PCPs I, including those from mammals. The residues from other loops also contribute significantly towards pG binding. The <scene name='80/806393/Cv/15'>main chain nitrogen and oxygen of Leu71 form two hydrogen bonds with OE and NH of pG</scene>, respectively. The adjacent residue, <scene name='80/806393/Cv/16'>Gly70</scene>, forms van der Waals contacts with OE of pG. Other van der Waals contacts for the pG binding are formed by the side chains of <scene name='80/806393/Cv/17'>Tyr142, Val143, Cys144 and Val45</scene>. The 5' carboxylate group of pG is held by several ionic interactions: <scene name='80/806393/Cv/18'>one of its carboxylate oxygen forms a hydrogen bond with NE2 of His169</scene> while the <scene name='80/806393/Cv/19'>other carboxylate oxygen interacts with the main chain N of catalytic Cys144 and a water molecule</scene>. This water molecule is further stabilized by its interactions with the residues Tyr142, Asn145, and Ala139. | ||
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| - | diagram of PCPdr monomer (spectrum color) is shown in cartoon with pG bound to the active site shown in magenta sticks. (c) Active-site of PCPdr with pG binding residues shown in sticks and bound pG is shown in magenta sticks. (d) LigPlot illustration of the active site showing various interactions with pG residue. (Wallace et. al. 1996). | ||
| - | Figure 2 Enzyme Kinetics of PCPdr. Activity towards pG-BNA substrate was tested at various substrate concentrations at 50˚C and pH 8. The Km was calculated by curve fitting to Michaelis Menten model using Graph Pad Prism software (ver. 6.0). | ||
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| - | Figure 3 Electron density maps of PCPdr active site. Stereo view of electron density maps of PCPdr active site around pG binding site. Fourier map (2mFo-DFc) shown in gray color contoured at 2.0 sigma around key residues of the active site. Simulated-annealing omit map (green color) contoured at 4.0 sigma showing electron density for pG residue (magenta sticks). | ||
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<scene name='80/806393/Cv1/10'>Structural conservation of loop-A in the crystal structures of PCPs from different prokaryotes</scene>. PCPdr (present study; gray); Thermus thermophlius (PDB entry 2ebj; orange); Pyrococcus furiosus (PDB entry 2df5; violet); Thermococcus litoralis (PDB entry 1a2z; marine blue); Pyrococcus horikoshii (PDB entry 1iu8; yellow); Bacillus amyloliquefaciens (PDB entry 1aug; lime green); Bacillus anthrasis (PDB entry 3lac; chocolate) Xenorhabdus bovienii (PDB entry 4gxh; brown) and Staphylococcus aureus (PDB entry 3giu; cyan). (Value in the bracket is reference of Bacillus amyloliquefaciens). | <scene name='80/806393/Cv1/10'>Structural conservation of loop-A in the crystal structures of PCPs from different prokaryotes</scene>. PCPdr (present study; gray); Thermus thermophlius (PDB entry 2ebj; orange); Pyrococcus furiosus (PDB entry 2df5; violet); Thermococcus litoralis (PDB entry 1a2z; marine blue); Pyrococcus horikoshii (PDB entry 1iu8; yellow); Bacillus amyloliquefaciens (PDB entry 1aug; lime green); Bacillus anthrasis (PDB entry 3lac; chocolate) Xenorhabdus bovienii (PDB entry 4gxh; brown) and Staphylococcus aureus (PDB entry 3giu; cyan). (Value in the bracket is reference of Bacillus amyloliquefaciens). | ||
Revision as of 11:08, 27 January 2019
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