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The catalytic architecture of leukotriene C4 synthase with two arginine residues.

Saino H, Ukita Y, Ago H, Irikura D, Nisawa A, Ueno G, Yamamoto M, Kanaoka Y, Lam BK, Austen KF, Miyano M - J. Biol. Chem. (2011)

Bottom Line: LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH).A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104.In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model.

View Article: PubMed Central - PubMed

Affiliation: Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, Sayo, Hyogo, Japan.

ABSTRACT
Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).

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Difference Fourier maps of the R31A and R104A                                        mutants. The difference Fourier electron density maps                                    for R31A (panel A) and R104A (panel                                        B) were calculated from Fourier coefficients and                                    the phases of FR31A(F23) −                                    FWT(F23), φwt(F23) and                                        FR104A(C2221) - FWT(C2221),                                        φwt(C2221), respectively. The                                        R-factors between the diffraction intensity                                    data of the WT LTC4S and the R31A mutant and between                                    the diffraction intensity data of the WT LTC4S and                                    the R104A mutant were 0.082 and 0.192, respectively. The map for                                    R31A was contoured at +4.5σ                                    (blue) and −4.5σ                                        (red), and that for R104A was contoured at                                    +4σ (blue) and −4σ                                        (red). The atomic models in panel                                        A and B are the WT                                    LTC4S in this work and the previous work (PDB ID                                        2PNO), respectively.
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Figure 5: Difference Fourier maps of the R31A and R104A mutants. The difference Fourier electron density maps for R31A (panel A) and R104A (panel B) were calculated from Fourier coefficients and the phases of FR31A(F23) − FWT(F23), φwt(F23) and FR104A(C2221) - FWT(C2221), φwt(C2221), respectively. The R-factors between the diffraction intensity data of the WT LTC4S and the R31A mutant and between the diffraction intensity data of the WT LTC4S and the R104A mutant were 0.082 and 0.192, respectively. The map for R31A was contoured at +4.5σ (blue) and −4.5σ (red), and that for R104A was contoured at +4σ (blue) and −4σ (red). The atomic models in panel A and B are the WT LTC4S in this work and the previous work (PDB ID 2PNO), respectively.

Mentions: There were no significant residual electron densities, except for the residual electron density corresponding to the side chain of the mutated amino acid residue in both of the difference Fourier electron density maps for each pair of the isomorphous crystals (Fig. 5). The difference Fourier electron density maps supported the conclusion that the three-dimensional structures of the R31A and R104A mutants did not suffer any damage from the point mutations.


The catalytic architecture of leukotriene C4 synthase with two arginine residues.

Saino H, Ukita Y, Ago H, Irikura D, Nisawa A, Ueno G, Yamamoto M, Kanaoka Y, Lam BK, Austen KF, Miyano M - J. Biol. Chem. (2011)

Difference Fourier maps of the R31A and R104A                                        mutants. The difference Fourier electron density maps                                    for R31A (panel A) and R104A (panel                                        B) were calculated from Fourier coefficients and                                    the phases of FR31A(F23) −                                    FWT(F23), φwt(F23) and                                        FR104A(C2221) - FWT(C2221),                                        φwt(C2221), respectively. The                                        R-factors between the diffraction intensity                                    data of the WT LTC4S and the R31A mutant and between                                    the diffraction intensity data of the WT LTC4S and                                    the R104A mutant were 0.082 and 0.192, respectively. The map for                                    R31A was contoured at +4.5σ                                    (blue) and −4.5σ                                        (red), and that for R104A was contoured at                                    +4σ (blue) and −4σ                                        (red). The atomic models in panel                                        A and B are the WT                                    LTC4S in this work and the previous work (PDB ID                                        2PNO), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3091245&req=5

Figure 5: Difference Fourier maps of the R31A and R104A mutants. The difference Fourier electron density maps for R31A (panel A) and R104A (panel B) were calculated from Fourier coefficients and the phases of FR31A(F23) − FWT(F23), φwt(F23) and FR104A(C2221) - FWT(C2221), φwt(C2221), respectively. The R-factors between the diffraction intensity data of the WT LTC4S and the R31A mutant and between the diffraction intensity data of the WT LTC4S and the R104A mutant were 0.082 and 0.192, respectively. The map for R31A was contoured at +4.5σ (blue) and −4.5σ (red), and that for R104A was contoured at +4σ (blue) and −4σ (red). The atomic models in panel A and B are the WT LTC4S in this work and the previous work (PDB ID 2PNO), respectively.
Mentions: There were no significant residual electron densities, except for the residual electron density corresponding to the side chain of the mutated amino acid residue in both of the difference Fourier electron density maps for each pair of the isomorphous crystals (Fig. 5). The difference Fourier electron density maps supported the conclusion that the three-dimensional structures of the R31A and R104A mutants did not suffer any damage from the point mutations.

Bottom Line: LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH).A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104.In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model.

View Article: PubMed Central - PubMed

Affiliation: Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, Sayo, Hyogo, Japan.

ABSTRACT
Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).

Show MeSH
Related in: MedlinePlus