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Soybean lipoxygenase isoenzyme L3 By 관리자 / 2018-07-13 AM 11:17 / 조회 : 853회
Soybean lipoxygenase isoenzyme L3 Soybean lipoxygenase isoenzyme L3 represents a second example (after L1) of the X-ray structure (R=17% at 2.6Å resolution) for a member of the large family of lipoxygenases. L1 and L3 have different characteristics in catalysis, although they share 72% sequence identity (the changes impact 255 amino acids) and similar folding (average C ( r.m.s.d. of 1Å). The critical non-heme iron site has the same features as for L1: 3O and 3N in pseudo C3v orientation, with two oxygens (from Asn713 and water) at a non-binding distance. Asn713 and His518 are strategically located at the junction of three channels connecting the cavity around the iron site with the molecule surface. The most visible differences between L1 and L3 isoenzymes occur in and near these channels, affecting their accessibility and volume. Among the L1/L3 substitutions Glu256/Thr274, Tyr409/His429 and Ser747/Asp766 affect the salt bridges (L1: Glu256…His248 and Asp490…Arg707) that in L1 restrict the access to the iron site from two opposite directions. The L3 molecule has a passage going through the whole length of the helical domain, starting at the interface with the Nt-domain (near 25-27 and 254-278) and going to the opposite end of the Ct-domain (near 367, 749). His:266, 513, 776, Phe:264, 272, 576, 714, Trp519, Arg:552, 726, Asp:766, 779 and Lys278 might be of importance for catalysis, as the likely candidates for the catalytic bases, possible aromatic radicals or amino acids assisting in oxygen transport or solvent withdrawal. The L3 structure and electron density map indicate that the salt bridge partitioning channel II (Arg726…Asp509)can be open, when an alternative rotamer of Arg726 forms a salt bridge with Asp766 (or Glu367), leaving Asp509 from a former ion pair to interact with His429. The residues Asp766 and His429 are sequential changes in relation to L1.The presence of channel III, possible opening of channel II and, in general, more room in the passages for substrate/product, provide a possible explanation for a stringent stereospecificity of catalytic products in L1 (that produces predominantly 13-hydroperoxide) versus the lack of such specificity in L3 (that turns out a mixture of 9- and 13-hydroperoxides and their diastereoisomers). Keywords: metalloprotein, lipoxygenase, X-ray structure, fatty acid oxydation Ewa Skrzypczak-Jankun, L. Mario Amzel), Beth Kroa, Max O. Funk, Jr. University of Toledo and )John Hopkins University PDB entry 1LNH “Structural and Thermochemical Characterization of Lipoxygenase Catechol Complexes” – Chau Pham, Jerzy Jankun, Ewa Skrzypczak-Jankun, Robert A. Flowers II and Max O. Funk Jr. Biochemistry (1998) A complex between native, iron(II), soybean lipoxygenase 3 and 4-nitrocatechol has been detected by isothermal titration calorimetry and characterized by X-ray crystallography. The compound moors in the central cavity of the protein, close to the essential iron atom, but not in the bonding arrangement with it. The iron ligands experience a significant rearangement upon formation of the complex relative to their positions in the native enzyme: a water molecule becomes bound to iron in the complex and one histidine ligand (His 518) moves away from the iron to become in the hydrogen bonding interaction with the catechol. Positional changes in residues cause the geometrical change in the iron coordination into a trigonal pyramid. Stoichiometry calculated from isothermal titration calorymetry shows fractional numbers indicating that catechol complexing process is highly dynamic and strongly depends upon oxidation state of the metal cofactor (0.4 in Fe(+2), and 1.3 in Fe(+3) complexes respectively). Molecular modeling and force field calculations allow to predict more than one binding site but conformed the result from x-ray analysis as energetically most favorable. These observation reveal specific details of the interaction between lipoxygenase and the catechol and raise the possibility that changes in the ligand environment of the iron atom could be a feature of the product activation reaction or the catalytic mechanism. 관련사이트 : http://golemxiv.dh.meduohio.edu/~ewa/research.html

Soybean lipoxygenase isoenzyme L3 By 관리자 / 2018-07-13 AM 11:17 / 조회 : 853회

Soybean lipoxygenase isoenzyme L3

Soybean lipoxygenase isoenzyme L3 represents a second example (after L1) of the X-ray structure (R=17% at 2.6Å resolution) for a member of the large family of lipoxygenases. L1 and L3 have different characteristics in catalysis, although they share 72% sequence identity (the changes impact 255 amino acids) and similar folding (average C ( r.m.s.d. of 1Å). The critical non-heme iron site has the same features as for L1: 3O and 3N in pseudo C3v orientation, with two oxygens (from Asn713 and water) at a non-binding distance. Asn713 and His518 are strategically located at the junction of three channels connecting the cavity around the iron site with the molecule surface. The most visible differences between L1 and L3 isoenzymes occur in and near these channels, affecting their accessibility and volume. Among the L1/L3 substitutions Glu256/Thr274, Tyr409/His429 and Ser747/Asp766 affect the salt bridges (L1: Glu256…His248 and Asp490…Arg707) that in L1 restrict the access to the iron site from two opposite directions. The L3 molecule has a passage going through the whole length of the helical domain, starting at the interface with the Nt-domain (near 25-27 and 254-278) and going to the opposite end of the Ct-domain (near 367, 749). His:266, 513, 776, Phe:264, 272, 576, 714, Trp519, Arg:552, 726, Asp:766, 779 and Lys278 might be of importance for catalysis, as the likely candidates for the catalytic bases, possible aromatic radicals or amino acids assisting in oxygen transport or solvent withdrawal. The L3 structure and electron density map indicate that the salt bridge partitioning channel II (Arg726…Asp509)can be open, when an alternative rotamer of Arg726 forms a salt bridge with Asp766 (or Glu367), leaving Asp509 from a former ion pair to interact with His429. The residues Asp766 and His429 are sequential changes in relation to L1.The presence of channel III, possible opening of channel II and, in general, more room in the passages for substrate/product, provide a possible explanation for a stringent stereospecificity of catalytic products in L1 (that produces predominantly 13-hydroperoxide) versus the lack of such specificity in L3 (that turns out a mixture of 9- and 13-hydroperoxides and their diastereoisomers).

Keywords: metalloprotein, lipoxygenase, X-ray structure, fatty acid oxydation

Ewa Skrzypczak-Jankun, L. Mario Amzel*), Beth Kroa, Max O. Funk, Jr.

University of Toledo and *)John Hopkins University

PDB entry 1LNH

“Structural and Thermochemical Characterization of Lipoxygenase Catechol Complexes” – Chau Pham, Jerzy Jankun, Ewa Skrzypczak-Jankun, Robert A. Flowers II and Max O. Funk Jr. Biochemistry (1998)

A complex between native, iron(II), soybean lipoxygenase 3 and 4-nitrocatechol has been detected by isothermal titration calorimetry and characterized by X-ray crystallography. The compound moors in the central cavity of the protein, close to the essential iron atom, but not in the bonding arrangement with it. The iron ligands experience a significant rearangement upon formation of the complex relative to their positions in the native enzyme: a water molecule becomes bound to iron in the complex and one histidine ligand (His 518) moves away from the iron to become in the hydrogen bonding interaction with the catechol. Positional changes in residues cause the geometrical change in the iron coordination into a trigonal pyramid. Stoichiometry calculated from isothermal titration calorymetry shows fractional numbers indicating that catechol complexing process is highly dynamic and strongly depends upon oxidation state of the metal cofactor (0.4 in Fe(+2), and 1.3 in Fe(+3) complexes respectively). Molecular modeling and force field calculations allow to predict more than one binding site but conformed the result from x-ray analysis as energetically most favorable. These observation reveal specific details of the interaction between lipoxygenase and the catechol and raise the possibility that changes in the ligand environment of the iron atom could be a feature of the product activation reaction or the catalytic mechanism.

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