Article Figures & Data
Figures
- Fig. 1.
Characterization of hAOX1-WT and the protein derived from a codon-optimized gene sequence (co-hAOX). (A) Size-exclusion chromatogram (absorbance at 450 nm), UV/Vis spectrum, and a 10% SDS-polyacrylamide gel of purified hAOX-WT. (B) Size-exclusion chromatogram (absorbance at 450 nm) and a 10% SDS-polyacrylamide gel of purified co-hAOX expressed using the codon-optimized gene sequence. The UV-Vis spectra were determined using purified air-oxidized hAOX1 proteins in 50 mM Tris (pH 8.0) at RT. The elution profiles of the size-exclusion chromatography in 50 mM Tris, 200 mM NaCl, and 1 mM EDTA, pH 8.0, on a Superdex 200 column show different peaks of wild-type hAOX1 protein corresponding to multimeric (600 kDa), dimeric (300 kDa), and monomeric (150 kDa) forms. The majority of the lower bands were identified by mass spectrometry as degradation products of hAOX1 (data not shown).
- Fig. 2.
Location of the amino acid exchanges in the hAOX structure as a result of SNPs identified in the hAOX1 gene. Close-up of the hAOX active site (PDB ID: 4UHW). Highlighted is the localization of the three SNPs discussed in this paper: Cys44 in pink, Gly1269 in green, and Ser1271 in orange. The Moco and the two FeS centers (and the corresponding Cys residues of FeSII: Cys49, Cys52 and Cys74) are also represented, as well as residues Glu1270 and Gln 1203. The figure was prepared using PyMOL v1.7.2 (Schrödinger, Cambridge, MA).
- Fig. 3.
Characterization of hAOX1 SNP. Size-exclusion chromatogram (absorbance at 450 nm), UV/Vis spectrum, and a 10% SDS-polyacrylamide gel of the variants (A) hAOX-S1271L, (B) hAOX-G1269R, and (C) hAOX1-C44W. The UV/Vis spectra were determined using purified air-oxidized hAOX1 proteins in 50 mM Tris (pH 8.0) at RT. The elution profiles of size-exclusion chromatography in 50 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0 on a Superdex 200 column show different peaks of hAOX variants corresponding to the multimeric (600 kDa), dimeric (300 kDa), and monomeric (150 kDa) forms.
- Fig. 4.
pH optimum of hAOX1-WT and hAOX1-S1271L. The pH optimum was determined for (A) hAOX1-WT and (B) hAOX1-S1271L by measuring activities of hAOX1 with phenanthridine as substrate and molecular oxygen as electron acceptor, in the pH range of 6.5–10 in 50 mM Tris/HCl buffer. Activity measurements were performed at RT.
- Fig. 5.
Lineweaver-Burk plots of hAOX1 inhibition by DCPIP. Inhibition of hAOX1 using DCPIP concentrations between 0 and 200 μM were measured: 200 μM (diamond), 100 μM (filled square), 50 μM (triangle), 10 μM (empty circle), 1 μM (empty square), and 0 μM (filled circle). Inset: secondary plot of 1/Vmaxapp and inhibitor concentration to determinate Ki value. The product formation was measured with 5–100 μM cinnamaldehyde as substrate using molecular oxygen as electron acceptor.
- Fig. 6.
Lineweaver-Burk plots of the inhibition of hAOX1-WT and variant hAOX1-S1271L by raloxifene. Lineweaver-Burk plots of hAOX1-WT (A) and hAOX1-S1271L (B) inhibition by raloxifene. Inhibition of human AOX1s using raloxifene concentrations between 0 and 250 nM were measured: 250 nM (square), 100 nM (empty circle), 25 nM (filled circle), 5 nM (triangle), and 0 nM (diamond). Inset: secondary plot inhibitor concentration to determinate Ki value. Phenanthridine as substrate and molecular oxygen as electron acceptor were used. Assays were performed at 25°C in Tris 50 mM, NaCl 200 mM, and 1 mM EDTA buffer, pH 8.0. Data are mean values from three independent measurements (± S.D.).
- Fig. 7.
Crystal structure of the hAOX1-S1271L variant. Close up of the hAOX1-S1271L variant active site. (A) Details of Leu1271 (orange) immediately after the catalytic Glu1270 residue are shown with the 2mFo–DFc map contoured at 1σ. (B) Comparison of hAOX1-S1271L (blue) and the hAOX1-WT (Coelho et al., 2015) for the Leu1271 and Ser1271 residue, respectively. Also shown is the localization of the G1269 SNP (green) and the Moco and FeSI cofactors. The figure was created using PyMOL v1.7.2.
Tables
hAOX1-S1271La Data collection Space group P42212 Cell dimensions a = b, c (Å) 147.40, 131.70 Resolution (Å) 49.15–3.41 (3.68–3.41) Rmerge 0.30 (1.75) I / s(I) 9.50 (2.20) Completeness (%) 100.00 (100.00) Redundancy 10.8 (11.1) Refinement Resolution (Å) 3.41 No. of reflections 20403 (4115) Rwork / Rfree 18.7 / 24.6 No. of atoms Protein 9995 Ligand/ion 13 Water 52 B-factors Protein 73.90 Ligand/ion 73.70 Water 35.60 RMS deviations Bond lengths (Å) 0.014 Bond angles (°) 1.545 RMS, root-mean-square deviation.
↵a Values in parentheses are for highest-resolution shell
- TABLE 2
Characterization of purified hAOX1-WT, the codon-optimized co-hAOX1 protein, and hAOX1 variants C44W, S1271L, and G1269R after expression in E. coli TP1000 cells
hAOX1-WT co-hAOX1 hAOX1- S1271L hAOX1- C44W hAOX1- G1269R Total yield of protein (mg/l of culture) 0.09 ± 0.02 1.1 ± 0.3 0.9 ± 0.1 ≥0.0005 1.2 ± 0.3 Specific activitya (mU/mg) of purified enzyme w/o in vitro chemical sulfuration 113.6 ± 34.1 141.4 ± 29.8 126.7 ± 21 — — Specific activitya (mU/mg) of purified enzyme after in vitro chemical sulfuration 1516.7 ± 182.7 1459.1 ± 170.5 1132.1 ± 155.3 — — Mo content (%)b 55.2 ± 8.41 66.4 ± 6.58 41.3 ± 3.54 — 31.2 ± 3.43 Fe content (%)b 71.2 ± 10.37 76.7 ± 5.63 58.6 ± 4.21 — 72.3 ± 4.66 ↵a Specific activity was determined using 40 μM phenanthridine as substrate and molecular oxygen as electron acceptor.
↵b Molybdenum and iron content were determined by inductively coupled plasma optical emission spectrometry (ICP-OES). Values are related to the theoretical 100% full complement of Moco and the 2× [2Fe2S] clusters.
- TABLE 3
Steady-state kinetics of hAOX1-WT and SNP variant S1271L
Steady-state kinetic parameters were calculated and related to a molybdenum saturation of 100% for hAOX1-WT and hAOX1-S1271L under assay conditions using molecular oxygen (phenanthridine), 1 mM ferricyanide, and 100 μM 2,6 dichlorophenolindophenol as electron acceptors. Data are mean values from three independent measurements (±S.D.).
Enzyme Substrate Electron Acceptor kcat KM kcat/KM min−1 μM min−1 μM−1 hAOX1-WT codon-optimized Phenanthridine Molecular oxygen 306.5 ± 11.7 15.91 ± 2.9 19.3 Ferricyanide 220.5 ± 5.6 24.85 ± 3.5 8.8 DCPIP 27.5 ± 5.0 1.8 ± 0.5 15.3 Phthalazine Ferricyanide 271.7 ± 13.9 196.2 ± 10.8 1.4 DCPIP 27.4 ± 3.7 22.14 ± 4.1 1.2 Benzaldehyde Ferricyanide 187.8 ± 13.9 80.16 ± 8.4 2.3 DCPIP 56.9 ± 8.6 12.1 ± 2.7 4.7 hAOX1-WT native sequence (pSS130) Phenanthridine Molecular oxygen 284.8 ± 11.3 26.53 ± 6.5 10.7 Ferricyanide 292.1 ± 15.8 25.5 ± 4.8 11.5 DCPIP 26.8 ± 1.4 0.78 ± 0.1 34.4 Phthalazine Ferricyanide 223.3 ± 10.6 125.7 ± 4.1 1.8 DCPIP 31.62 ± 3.9 8.96 ± 0.8 3.5 Benzaldehyde Ferricyanide 203.7 ± 12.1 45.18 ± 6.2 4.5 DCPIP 29.8 ± 1.1 4.6 ± 0.3 6.5 hAOX1-S1271L Phenanthridine Molecular oxygen 233.36 ± 13.6 12.03 ± 0.9 19.4 Ferricyanide 124.96 ± 2.5 15.98 ± 3.9 7.8 DCPIP 15.65 ± 1.3 1.56 ± 0.1 10.0 Phthalazine Ferricyanide 175.6 ± 8.2 177.4 ± 12.4 0.9 DCPIP 17.44 ± 0.7 12.16 ± 0.8 1.4 Benzaldehyde Ferricyanide 95.7 ± 5.2 67.8 ± 10.9 1.4 DCPIP 15.46 ± 1.2 7.83 ± 0.7 1.9
In this issue
Kinetic Characterization of SNP-Based Variants of Human AOX1
