Fig 1.
Steady-state kinetic parameters for mAOX1-4 with aromatic aldehydes as substrates containing a benzyl-group.
Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the KM. The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Fig 2.
Steady-state kinetic parameters for mAOX1-4 with N-heterocyclic compounds as substrates.
Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the KM. The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Fig 3.
Steady-state kinetic parameters for mAOX1-4 with aliphatic aldehydes as substrates.
Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the KM. The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Fig 4.
Steady-state kinetic parameters for mAOX1-4 with cinnamaldehyde-related compounds as substrates aromatic aldehydes as substrates.
Apparent steady-state kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. The substrate concentrations were varied around 0.5 and 10 times the KM. The chemical structure of each substrate is shown in the Fig. The values were corrected to a molybdenum saturation of 100% for each mAOX variant for a better comparability. Kinetic Data are mean values from three independent measurements (±S.D.). n.d. = no activity detectable.
Fig 5.
Active site and substrate-binding tunnel in mAOX4.
Shown is a model of the structure of mAOX4 active site generated with Pymol using hAOX1 as template. Shown is the surface-representation of the substrate-binding site of mAOX4 with highlighted residues in the conserved region of the active site and in the non-conserved region which dictate substrate specificity. The substrate-funnel is highlighted in blue according to the study by Cerqueira et al. [20]. Residues in red indicate the amino acids whose nature only specific for mAOX4. Residues in blue are directly involved in substrate binding or in initiating the catalytic mechanism. The Moco and FeSI is shown in stick representation.
Table 1.
Comparison of the active site amino acid residues of hAOX1, mAOX1, mouse AOX3, mAOX4, mAOX2, and bXDH which were shown to be specific to mAOX4 [20].
Fig 6.
UV-Vis spectra of mAOX4 variants in comparison to mAOX4-wild-type.
The Fig illustrates UV-Vis spectra of 10 μM mAOX4 wild-type and variants (as indicated) in the oxidized state recorded in 50 mM Tris, pH 8.0.
Fig 7.
Molybdenum and iron saturation of purified mAOX4 variants.
After purification, the molybdenum and iron content of the indicated enzymes were determined by inductively coupled plasma optical emission spectroscopy. The iron content corresponds to saturation with both, FeSI and FeSII clusters (corresponding to four molecules of Fe). The 100% values are set to a full saturation of the enzyme with of Moco and 2x[2Fe-2S] clusters.
Table 2.
Steady state kinetic parameters mAOX4 wildtype and variants with different substrates.
Steady-state kinetics were corrected to molybdenum saturation of 100%. Kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. Substrate concentration were varied around 0.5 and 10 times the KM. Data are mean values from three independent measurements (±S.D.).