PMC

Schematic depicting reactions catalyzed by sepiapterin reductase. Upper panel, reduction of sepiapterin by sepiapterin reductase generates dihydrobiopterin. Additional cellular reductases convert dihydrobiopterin to tetrahydrobiopterin. Lower panel, in the presence of redox cycling chemicals such as menadione, sepiapterin reductase generates reactive oxygen species.

Effects of N-acetylserotonin and dicoumarol on sepiapterin reduction and redox cycling by human recombinant sepiapterin reductase. Sepiapterin reductase activity was measured by decreases in sepiapterin absorbance at 420 nm. Redox cycling was measured by the formation of superoxide anion in enzyme assays in the presence of 100 μm menadione. Note that both N-acetylserotonin and dicoumarol inhibit sepiapterin reduction (IC₅₀ = 2.6 and 0.2 μm, respectively) but not redox cycling activity. Sepiapterin reduction was analyzed by changes in absorbance of sepiapterin at 420 nm.

Inhibition of dihydrobiopterin and tetrahydrobiopterin formation by menadione in A549 lung epithelial cells. Cells were pretreated with control medium or medium containing 500 μm menadione. After 2 h, sepiapterin (100 μm final concentration) was added to the cultures. After an additional 2 h, cells were extracted and analyzed for BH₂ and BH₄ content by HPLC. The upper and middle tracings were from cells treated with sepiapterin alone or sepiapterin and menadione. The lower tracing is from control cells that were not treated with sepiapterin or menadione.

Oxygen consumption by human recombinant sepiapterin reductase during chemical redox cycling. A Clark-type oxygen electrode was used to quantify oxygen consumption during redox cycling. The reaction was run in the presence of 200 μm NADPH and an NADPH-regenerating system. After establishing a stable base line, SPR or sepiapterin was added. Panels A and B, 5 μm 9,10-phenanthrenequinone (9,10-PQ), but not sepiapterin (200 μm), initiated oxygen consumption by sepiapterin reductase. Panels C and D, 200 μm phenylquinone, but not 200 μm N-acetylserotonin, inhibited 9,10-phenanthrenequinone-induced oxygen consumption.

Molecular models of sepiapterin reductase complexed with NADP⁺ and sepiapterin. Panel A, superposition of human and mouse sepiapterin reductase. Human sepiapterin reductase is shown in violet (Protein Data Bank code 1Z6Z) and mouse sepiapterin reductase in cyan (Protein Data Bank code 1SEP). The NADP cofactor and sepiapterin substrate complexed with mouse sepiapterin reductase are shown in red and green, respectively. NADP bound with human sepiapterin reductase is shown in yellow. Panel B, close-up of the active site of the NADP-sepiapterin-sepiapterin reductase complex. Key residues in the active site are represented as blue sticks. Panel C, interactions of sepiapterin and NADP with the active site of human sepiapterin reductase. Protein residues selected for site-directed mutagenesis are shown as blue sticks. Hydrogen bonds are shown by broken lines, and the corresponding distances (Å) are indicated.

Effects of quinones on human recombinant sepiapterin reductase activity. Panel A, effects of quinones (9,10-phenanthrenequinone (9,10-PQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), menadione (MD), and dimethoxy-1,4-naphthoquinone (DMNQ)) on sepiapterin (SP) reduction by human recombinant sepiapterin reductase. Panel B, Lineweaver-Burk analysis of menadione inhibition of sepiapterin reduction. Note that menadione is a noncompetitive inhibitor. Panels A and B, sepiapterin reduction was measured by changes in absorbance of sepiapterin at 420 nm. Panel C, correlation between the k_cat for redox cycling, as measured by H₂O₂ production, and the ability to inhibit reduction of sepiapterin. Data are presented as the IC₅₀ value for inhibition of sepiapterin reduction. Panel D, correlation between k_cat/K_m for redox cycling by sepiapterin reductase and redox potential for various quinones.

Ability of human recombinant sepiapterin reductase to generate ROS. Reaction mixes contained sepiapterin reductase, 200 μm NADPH, and 500 μm menadione. Panel A, menadione stimulated superoxide anion production by sepiapterin reductase. Menadione was added to reaction mixes to stimulate superoxide anion formation as indicated by the arrow. Superoxide anion was measured spectrophotometrically by monitoring superoxide dismutase inhibitable changes in absorbance of acetylated cytochrome c at 550 nm. Reactions were run in the absence and presence of SPR. SOD (40 units), which dismutates superoxide anion, was added to the reactions as indicated by the arrowheads. Panel B, menadione stimulated H₂O₂ production by sepiapterin reductase. Menadione was added to reaction mixes to stimulate H₂O₂ production. Catalase (2000 units), which breaks down H₂O₂, was added to the reactions as indicated by the arrowheads.

Characterization of redox cycling by human recombinant sepiapterin reductase. Panel A, redox cycling of different quinones by sepiapterin reductase. H₂O₂ formation was measured in the absence or presence of 5 μm 9,10-phenanthrenequinone, 500 μm menadione, or 500 μm dimethoxy-1,4-naphthoquinone. Data are the average of three independent measurements. SPR was added at the indicated time. Panel B, effects of increasing concentrations of NADPH on menadione redox cycling; inset, effects of increasing concentrations of menadione on redox cycling activity. Panel C, inhibition of menadione redox cycling by 100 μm benzoquinone or phenylquinone; inset, inability of benzoquinone and phenylquinone to redox cycle with sepiapterin reductase. Panel D, effects of benzoquinone and phenylquinone on sepiapterin reductase activity. Enzyme activity was analyzed by changes in absorbance of sepiapterin at 420 nm.

Effects of oxygen on H₂O₂ production by 9,10-phenanthrenequinone-induced redox cycling with sepiapterin reductase. Reactions were run in Oxygraph reaction cells in a total volume of 0.6 ml and contained 50 mm phosphate buffer, pH 7.8, 5 μm 9,10-phenanthrenequinone, and 200 μm NADPH. To generate reduced oxygen levels, the reaction cell was purged with 100% nitrogen gas, and levels of oxygen were monitored with the Oxygraph. To initiate the redox cycling reactions, 0.3 μg of sepiapterin reductase was injected into the reaction cells. Samples were removed at different time points; the reactions were stopped by the addition of acetonitrile at a final concentration of 30%, and the H₂O₂ concentration was determined by the Amplex red assay.

Site-directed mutagenesis of human recombinant sepiapterin reductase. Panel A, schematic drawing of recombinant sepiapterin reductase and mutant enzymes. The human recombinant sepiapterin reductase used in this investigation contains a hexahistidine tag at the N terminus and the 261 original full-length amino acids. The NADPH-binding motif, active center, and substrate transfer motif are indicated. The mutated positions are labeled separately. Panel B, sepiapterin reduction and redox cycling activity of sepiapterin reductase and mutant enzymes were assayed as described under “Experimental Procedures.” Data are presented as percentage of the wild type control enzyme activity. Sepiapterin reduction was assayed in the presence of 50 μm sepiapterin and measured by changes in absorbance of sepiapterin at 420 nm. Redox cycling was assayed in the presence of 500 μm menadione.

Identification of sepiapterin reductase as a mediator of chemical redox cycling. Panel A, redox cycling activity, assessed by the formation of H₂O₂, was quantified in 100,000 × g supernatant fractions from MLE-15 cells in the presence of 500 μm paraquat or diquat. Arrow indicates initiation of the reaction following the addition of supernatant. Panels B and C, paraquat-stimulated redox cycling activity in cytosolic fractions of MLE-15 cells purified by ADP affinity and size exclusion chromatography, respectively. Panel C, inset, SDS-PAGE analysis of total cell lysate and the two peaks (I and II) of redox cycling activity following size exclusion chromatography. The major band in peak II of SDS-PAGE (shown by the arrow) was analyzed by MALDI-TOF/TOF and identified as sepiapterin reductase. Panel D, dihydrobiopterin (BH₂) formation from sepiapterin by human recombinant sepiapterin reductase. Sepiapterin reductase activity required sepiapterin, NADPH, and purified recombinant SPR. Formation of BH₂ in enzyme assays was analyzed by HPLC. Inset, SDS-PAGE analysis of recombinant human sepiapterin reductase expressed in E. coli, lane 1, crude extract of E. coli containing sepiapterin reductase induced with 0.5 mm isopropyl β-d-thiogalactopyranoside; lane 2, sample collected from flow-through fraction from the Ni-NTA column; lane 3, sample of imidazole eluted fraction from nickel-affinity column. The arrow indicates the purified enzyme. Panel E, reduction of sepiapterin by purified recombinant sepiapterin reductase was dependent on NADPH, but not NADH. Inset, comparison of NADH and NADPH oxidation by sepiapterin reductase. Enzyme activity was analyzed by changes in absorbance of sepiapterin at 420 nm. Panel F, purified recombinant sepiapterin reductase mediates redox cycling of diquat.

Assays for sepiapterin reduction and redox cycling activity of sepiapterin reductase. Panel A, changes in the absorbance spectra of sepiapterin in the sepiapterin reductase assay. Note the decrease in absorption of sepiapterin over time. Standard reaction mixes in a total volume of 0.2 ml contained 50 mm phosphate buffer, pH 7.8, 200 μm NADPH, 50 μm sepiapterin, and 1 μg/ml sepiapterin reductase and were analyzed after 0 min (curve a), 1 min (curve b), 2 min (curve c), and 3 min (curve d). Controls contained the standard reaction mix without the enzyme and sepiapterin (curve e), with the standard reaction mix buffer plus 250 μm menadione (curve f), or with the standard reaction mix buffer plus 50 μm BH2 (curve g). Panel B, comparison of sepiapterin consumption and BH2 production in sepiapterin reductase enzyme assays. Both sepiapterin reduction reactions were run under the conditions indicated above. Sepiapterin reduction was measured by decreases in absorbance at 420 nm and was presented as micromolar concentrations of the substrate remaining in the assay over time. BH2 was measured using HPLC as described under “Experimental Procedures” and was presented as micromolar concentrations of the product formed in the assay over time. Panel C, effects of DTPA on H₂O₂ formation by sepiapterin reductase. Redox cycling was run in standard reaction mixes without sepiapterin and supplemented with 5 μm 9,10-phenanthrenequinone without (open triangles) and with 250 μm DTPA (closed triangles). In control experiments, sepiapterin reductase was left out of reaction mixes without (open circles) and with DTPA (closed circles). Panel D, comparison of H₂O₂ production by sepiapterin reductase redox cycling and autooxidation of BH2 and/or BH4. H₂O₂ production in the redox cycling reaction was compared with autooxidation of 50 μm BH2, 50 μm BH4, or the combination of BH2 and BH4. Autooxidation reactions were run in the buffer of the standard reaction mix. Inset, enlarged scale for autooxidation reactions run over 10 min. Note that the redox cycling reaction generated much greater amounts of H₂O₂ when compared with the autooxidation reactions.