13 research outputs found
The differences between the residues of coral cAOSs and <i>C</i>. <i>imbricata</i> cHPL.
<p><b>A</b>–alignment of amino acid sequences of coral cAOSs and <i>C</i>. <i>imbricata</i> cHPL presenting the conserved (blue) and distinct (grey vs red, respectively) residues in the substrate channel. The following amino acid sequences were compared: <i>Gersemia fruticosa</i> cAOS (NCBI ID: EU082210.1); <i>P</i>. <i>homomalla</i> cAOS (NCBI ID: AF003692.1); <i>Clavularia viridis</i> cAOS (NCBI ID: AB188528.1); <i>C</i>. <i>imbricata</i> cAOS (NCBI ID: KF000373.1); <i>C</i>. <i>imbricata</i> cHPL (NCBI ID: KF000374.1). <b>B–</b>the crystal structure of <i>P</i>. <i>homomalla</i> cAOS (blue) superpositioned with the model of <i>C</i>. <i>imbricata</i> cHPL (red) highlighting the main differences in the substrate pocket. The difference in the SSSAGE155-160PVKEGD fragments is not shown due to the illustrative purposes. The conserved amino acids between cAOS and cHPL are presented as a white and grey backbone, respectively. The heme is presented in yellow and the heme iron in orange.</p
The oligomerization analysis of <i>C</i>. <i>imbricata</i> protein samples.
<p><b>A</b>—wt cHPL; <b>B</b>—cHPL YS176-177NL. Similar oligomerization states were determined also for cHPL ME59-60LK, P65A, F150L. <b>C</b>—cHPL I357V; <b>D</b>—cHPL R56G; <b>E</b>—wt cHPL-LOX; <b>F</b>—wt cAOS; <b>G</b>—cAOS L150F. Oligomers and monomers of wt cHPL and mutants eluted at 8 and 16 min, respectively.</p
The docking analysis of <i>P</i>. <i>homomalla</i> cAOS with 8<i>R</i>-HpETE and AO.
<p><b>A</b>– 8<i>R</i>-HpETE located in the substrate pocket; <b>B</b>–the rotated view of 8<i>R</i>-HpETE in the substrate pocket with a hydrophobic surface; <b>C</b>–AO in the substrate pocket; <b>D–</b>an alternative positioning of AO in the substrate pocket. The colors used in the figure are presented as follows: heme–yellow; heme iron–orange; ligands–green; the interacting residues of <i>P</i>. <i>homomalla</i> cAOS–blue; the interacting residues of <i>C</i>. <i>imbricata</i> cHPL–cyan; residues supporting the coordination of ligand or heme–gray; oxygen atoms–red; nitrogen atoms–blue. For clarity, the distances between the ligand and selected residues are given in the text.</p
SDS-PAGE analysis of purified wt cHPL and corresponding mutants.
<p>Protein samples with an equal heme concentration (0.4 μM) were compared.</p
The products derived from the radiolabeled 8<i>R</i>-HpETE substrate by wt cHPL, selected cHPL mutants, and cAOS L150F.
<p><b>A</b>– C8-oxo acid formed by wt cHPL representing also the products formed by cHPL R56G, P65A, ME59-60LK, and SSSAGE155-160PVKEG; <b>B</b>– C8-oxo acid and α-ketol formed by cHPL F150L; <b>C</b>– C8-oxo acid, α-ketol and product 1 and product 2 formed by cHPL YS176-177NL; <b>D</b>–α-ketol formed by cAOS L150F. The product pattern is identical to wt cAOS’s (data not shown).</p
Structural and functional insights into the reaction specificity of catalase-related hydroperoxide lyase: A shift from lyase activity to allene oxide synthase by site-directed mutagenesis
<div><p>Two highly identical fusion proteins, an allene oxide synthase-lipoxygenase (AOS-LOX) and a hydroperoxide lyase-lipoxygenase (HPL-LOX), were identified in the soft coral <i>Capnella imbricata</i>. Both enzymes initially catalyze the formation of 8<i>R</i>-hydroperoxy-eicosatetraenoic acid (8<i>R</i>-HpETE) from arachidonic acid by the C-terminal lipoxygenase (LOX) domain. Despite the fact that the defined catalytically important residues of N-terminal catalase-related allene oxide synthase (cAOS) domain are also conserved in <i>C</i>. <i>imbricata</i> hydroperoxide lyase (cHPL), their reaction specificities differ. In the present study, we tested which of the amino acid substitutions around the active site of cHPL are responsible for a control in the reaction specificity. The possible candidates were determined via comparative sequence and structural analysis of the substrate channel and the heme region of coral cAOSs and <i>C</i>. <i>imbricata</i> cHPL. The amino acid replacements in cHPL—R56G, ME59-60LK, P65A, F150L, YS176-177NL, I357V, and SSSAGE155-160PVKEGD—with the corresponding residues of cAOS were conducted by site-directed mutagenesis. Although all these mutations influenced the catalytic efficiency of cHPL, only F150L and YS176-177NL substitutions caused a shift in the reaction specificity from HPL to AOS. The docking analysis of <i>P</i>. <i>homomalla</i> cAOS with 8<i>R</i>-HpETE substrate revealed that the Leu150 of cAOS interacts with the C5-C6 double bond and the Leu177 with the hydrophobic tail of 8<i>R</i>-HpETE. We propose that the corresponding residues in cHPL, Phe150 and Ser177, are involved in a proper coordination of the epoxy allylic radical intermediate necessary for aldehyde formation in the hydroperoxide lyase reaction.</p></div
Kinetic parameters of wt cHPL, wt cAOS and selected mutants with 8<i>R</i>-HpETE and H<sub>2</sub>O<sub>2</sub>.
<p>Kinetic parameters of wt cHPL, wt cAOS and selected mutants with 8<i>R</i>-HpETE and H<sub>2</sub>O<sub>2</sub>.</p
The proposed difference in reaction mechanisms between coral cAOS and cHPL.
<p>The hydrogen abstraction at C9 of the epoxy allylic carbocation is initiated by His67 of cAOS (blue). In cHPL-catalyzed reaction (red), no hydrogen abstraction occurs and instead, an unstable hemiacetal forms via the breakage of C8-C9 bond of the epoxide and the subsequent rebound of hydroxide.</p
RP-HPLC analysis of incubation products of C. <i>imbricata</i> tissue homogenate.
<p>A) Radio chromatogram of the products formed from [1<sup>−14</sup>C] AA by coral homogenate, extracted ion current (EIC) corresponding to α-ketol ([M<sup>−</sup>] = 335.2, peak 2), cyclopentenone ([M<sup>−</sup>] = 317.2, peak 3) and HETE ([M<sup>−</sup>] = 319.2, peak 3). B) The conversion of [1<sup>−14</sup>C] AA into unidentified polar compounds (UPC) (peak 1) and α-ketol (peak 2) in response to wounding. CPM - counts per minute.</p
List of primers: (a) degenerative primers used for isolation of the target genes, (b) primers used for 5′-3′ RACE and (c) qPCR primers used for gene expression analysis.
<p>Up - forward primer, down - reverse primer; all sequences are presented in the 5′ to 3′ direction.</p