Fig 1.
Desferrioxamine B biosynthetic pathway in S. sviceus.
Organization of the biosynthetic gene cluster in S. sviceus and proposed enzymatic steps involved in the synthesis of desferrioxamine B. Genes encoding the four desferrioxamine biosynthetic enzymes are in black (desA, desB, desC, and desD) and genes involved in ferrioxamine uptake and utilization are in grey (desE and desF).
Table 1.
Steady-state kinetic parameters determined in NAD(P)H oxidation assays for different substrates.
Fig 2.
Oxygraph traces demonstrate high oxidase activity in the absence of substrate. Assays contained 100 mM sodium phosphate buffer, pH 8 with 0.7 mM NADPH and 0.05 mM FAD in the absence or presence of 10 mM cadaverine.
Table 2.
Steady-state kinetic parameters determined in oxygen consumption assays for different substrates.
Fig 3.
Representative steady-state kinetics of SsDesB in oxygen consumption and product formation assays.
A. Initial rate kinetic data obtained with cadaverine and putrescine in oxygen consumption assays. Kinetic data for putrescine assayed with SsDesB, 0.05 mM FAD, and 0.7 mM NADPH. These data were obtained in triplicate and fit to the Haldane substrate inhibition (cadaverine) and Michaelis-Menten equations (putrescine), respectively. B. Initial rate data for cadaverine and putrescine assayed with SsDesB, 0.05 mM FAD, and 0.7 mM NADH in product formation assays. These data were obtained in triplicate and fit to the Haldane substrate inhibition (cadaverine) and Michaelis-Menten equations (putrescine), respectively.
Fig 4.
LC/MS detection of N-hydroxycadaverine (m/z 563.25).
LC/MS select ion chromatograms of Fmoc-derivatized N-hydroxycadaverine in assays with and without SsDesB. Diastereomers are observed.
Table 3.
Steady-state kinetic parameters determined in product formation assays with SsDesB and different substrates.
Fig 5.
Relative SsDesB activity with different substrates.
Initial rates measured with 10 mM L-lysine, cadaverine, spermidine, putrescine, n-butylamine, L-ornithine, 1,3-diaminopropane, and 3-dimethylaminopropylamine in product formation assays in the presence of SsDesB, 50 μM FAD, and 0.7 mM NADPH. All initial rates are relative to that of cadaverine. n-Butylamine, L-ornithine, 1,3-diaminopropane, and 3-dimethylaminopropylamine did not yield any Fmoc-derivatized N-hydroxylated products by LC/MS. All assays were performed in triplicate and molecular structures are shown with numbered carbon chains.
Fig 6.
Overall structure of tetrameric SsDesB in complex with NADP+.
NAD+ and FAD-bound SsDesB (PDB code: 6XBB). A) Tetrameric structure of SsDesB complexed with NADP+ illustrating the FAD-binding domain (red-ribbons) and the NADPH-binding domain (blue-ribbons). The bound NADP+ molecule is shown in orange spheres and the FAD molecule in yellow spheres. B) A view of the SsDesB protomer.
Fig 7.
Structural comparison of SsDesB and homologs.
Superimposed crystal structures of chain A of SsDesB (PDB code: 6XBC) with A) E. amylovora DfoA (PDB code: 5O8P), B) Kutzneria sp. 744 ornithine hydroxylase, Ktzl, (PDB code: 4TM1), C) P. aeruginosa ornithine hydroxylase, PvdA, (PDB code: 3S5W), and D) A. fumigatus ornithine hydroxylase, SidA, (PDB code: 5CKU).
Table 4.
Crystallographic data collection and refinement statistics.
Fig 8.
Active site environment of SsDesB holoenzyme.
View of the active site residues (carbon atoms in gray, nitrogen atoms in blue, and oxygen atoms in red) mediating FAD (carbon atoms in yellow, and phosphate atoms in orange) binding in the FAD-bound SsDesB structure (PDB code: 6XBC). The fit of the FAD molecule to the final 2Fo-Fc electron density map (blue mesh, 2.86 Å resolution, contoured at 1σ level) is shown.
Fig 9.
Active site environment of SsDesB-NADP+ bound structure.
Stereo-view of the active site of the SsDesB-NADP+ bound structure (PDB code: 6XBB). The fit of the FAD molecule (carbon atoms in yellow, nitrogen atoms in blue, oxygen atoms in red, and phosphate atoms in orange) and the NADP+ molecule (carbon atoms in orange) to the final 2Fo-Fc electron density map is shown (blue mesh, 2.37 Å resolution, contoured at 1σ level).
Fig 10.
Structural changes upon NADP+ binding.
View of the superimposed active sites of the SsDesB holoenzyme (blue; PDB code: 6XBC) and SsDesB-NADP+ bound (green; PDB code: 6XBB) structures highlighting the positional shift of residues in the active site upon NADP+ binding.
Fig 11.
General mechanism for Class B flavin-dependent monooxygenases.
The NADPH oxidation, oxygen consumption, and N-hydroxylation steps are represented by steps 1, 2, and 4, respectively. The loss of peroxide is indicated in blue and the apparent kcat values for steady-state kinetic assays with varied NADPH concentrations are shown. *, the substrate is either added before or after the reduction of molecular oxygen.
Fig 12.
Structural analysis of substrate specificity.
A. Active site structure of SidA bound to NADP+ and ornithine (PDB code: 4b63). B. Model of ornithine binding to the active site of SsDesB obtained by superimposing the coordinates of SsDesB (PDB code: 6XBB) onto the coordinates of SidA bound to ornithine (PDB code: 4b63). C. Comparison of the active site environment between SsDesB and SidA. D. Modeling of lysine binding to SsDesB. E. Modeling of cadaverine binding to SsDesB. F. Modeling of putrescine binding to SsDesB.