1. Volume 25, Issue 4, Pages 426-438.e4 (April 2018)
Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3- Monooxygenase 
Hyun Tae Kim, Byeong Kwan Na, Jiwoung Chung, Sulhee Kim, Sool Ki Kwon, Hyunju Cha, Jonghyeon Son, Joong Myung Cho, Kwang Yeon Hwang 
Cell Chemical Biology 
Volume 25, Issue 4, Pages e4 (April 2018)
DOI: /j.chembiol
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2. Cell Chemical Biology 2018 25, 426-438. e4DOI: (10. 1016/j. chembiol
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3. Figure 1 Comparison of the Active Site in PHBH and KMO
(A) Conformations of the flavin in PHBH. The isoalloxazine of the flavin is mobile and adopts “in,” “out,” and “open” conformations during catalysis. The conserved Tyr194 in human KMO blocks movement of the flavin in KMO (gray). The “in” (PDB: 1PBE), “out” (PDB: 1DOD), and “open” (PDB: 1K0L) positions of the flavin in PHBH are colored green, magenta, and orange, respectively.
(B) The active site in pfPHBH and pfKMO (PDB: 5NAK). pfPHBH (green) includes a distinct hydrogen bonding network in the active site that is absent in pfKMO (gray). L-Kyn, pOHB, and FAD are colored cyan, pink, and yellow, respectively.
(C) The mode of NADPH binding in substrate-free PHBH reflects an inactive conformation with the nicotinamide ring of NADPH located far away from the isoalloxazine ring of FAD. Superimposition of hKMO (gray) on Pseudomonas aeruginosa PHBH in complex with NADPH (blue; PDB: 1K0J). The red ellipse indicates the nicotinamide moiety of NADPH.
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4. Figure 2
Enzymatic Activity and Crystal Structure of hKMO Lacking the Transmembrane Domain Region
(A) Enzymatic activity of hKMO derivatives measured using LC-MS. The hKMO-446 protein, a deletion mutant of hKMO that includes the TMD region, is more active than the hKMO-FL, and was therefore used in activity assays. The hKMO-374 protein, which lacks the transmembrane domain (TMD) region, has no activity. Data are shown as mean ± SD.
(B) Crystal structure of the inactive form of human KMO (hKMO-374) lacking the TMD region. The C-terminal α helix is shown as a blue cylinder. The disordered region is represented as a red dotted line. FAD is shown in stick representation and colored according to atom type.
(C) Close-up view of the red box in (B) showing the substrate-binding site in the inactive KMO structure. Interactions between Asp358 and Arg85 and Tyr99 block substrate access. The disordered region is represented as a red dotted line.
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5. Figure 3 Structural Comparison of hKMO and scKMO
(A) Superimposition of the crystal structures of the inactive hKMO-374 (gray) and the active scKMO (red). The C-terminal α helix is shown as a cylinder.
(B) Close-up view of the blue box in (A) showing the positions of the F(F) residues in the loop located above the re side of the flavin.
(C) Superimposition of hKMO-374 and scKMO in complex with UPF-648 (green).
(D) Close-up of the red box in (C) showing the region surrounding the substrate-binding site. C-terminal α helix in scKMO in complex with UPF-648 is disordered.
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6. Figure 4
The Role of F(F) Loop Residues in Flavin Reduction by NADPH in KMO
(A) The F(F) residues are specific to KMOs. The sequence conservation cartoon representation of the S. cerevisiae KMO structure (PDB: 4J33) was produced using ConSurf (Goldenberg et al., 2009). Amino acids are colored based on sequence conservation grade as shown in the color-coding bar; from turquoise to maroon indicates variable to conserved residues, respectively. Active-site residues are highly conserved in all KMOs.
(B) Multiple sequence alignment of the active-site loop of KMOs from nine species and PHBH from P. fluorescens. The blue box represents the TG residues in PHBH that correspond to the F(F) loop residues (red box) in KMO.
(C) Sequence alignment of the loop region (residues 292–319) of human KMO with group A monooxygenases, which are designated by their UniProt accession numbers and colored cyan. The blue box indicates the sequence of human KMO. Residues corresponding to the F(F) loop residues in human KMO are highlighted in the red box. Multiple sequence alignment was performed using the Clustal Omega program on the UniProt website.
(D–F) Relative NADPH oxidase activity of (D) human, (E) S. cerevisiae, and (F) P. fluorescens KMO mutants. Data are shown as mean ± SD.
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7. Figure 5
NADPH Oxidation and H2O2 Production Assay in Human KMO, S. cerevisiae KMO, and P. fluorescens KMO
(A) Structures of the KMO inhibitors.
(B–D) Evaluation of the rates of NADPH oxidation and H2O2 production in the presence of L-Kyn, BA, UPF-648, or Ro in human KMO (B), S. cerevisiae KMO (C), and P. fluorescens KMO (D). Data are shown as mean ± SD.
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8. Figure 6
Comparison of the Binding Modes of Ro and UPF-648 in scKMO
(A) Superimposition of SF-scKMO (red) on Ro-scKMO (cyan). The C-terminal α helix is shown as a cylinder.
(B and C) The active site in Ro-scKMO. Interactions in the active site are shown as front (B) and top (C) views. F322 is colored red to show the position of this residue in SF-scKMO.
(D) Ro binding in the interface of scKMO does not cause conformational changes. The loop above the re side of the flavin is colored yellow. Domain II is shown in surface electrostatic representation.
(E) The structure of UPF-scKMO (PDB: 4J36). The C-terminal α helix is partially disordered due to UPF-648 binding.
(F) UPF-648 binding in the interface of scKMO causes conformational changes. The loop above the re side of the flavin is colored yellow. Domain II is shown in surface electrostatic representation.
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9. Figure 7 Structure of P. fluorescens KMO in Complex with Ro 61-8048
(A) Two binding sites for Ro are present in Ro-pfKMO. Domains I, II, and III are colored gray, green, and yellow, respectively.
(B) Superimposition of Ro complexed pfKMO (gray) and substrate-free pfKMO (blue). The red box indicates domain III.
(C) Rotation of the red box by 180°.
(D and E) Binding site I. Interactions in binding site I are shown as front (D) and top (E) views.
(F) Binding site II.
(G and H) Ro located in the interface between domains I and II of Ro-pfKMO (G) and Ro-scKMO (H). The loop above the re side of the flavin is colored yellow. Domain II is shown in surface electrostatic representation.
(I) Comparison of the binding modes of Ro in pfKMO (gray) and scKMO (magenta).
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