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Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from Acinetobacter baumannii

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  • معلومة اضافية
    • Publisher Information:
      American Chemical Society 2022-11-15
    • نبذة مختصرة :
      Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of A. baumannii has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an N-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by A. baumannii. FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the N-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (in vivo) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP+was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine N-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the N-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP+
    • الموضوع:
      Infectious Diseases; Acinetobacter baumannii; Cadaverine; Putrescine; NADP; Mixed Function Oxygenases; Flavins; Ornithine; Siderophores; Anti-Bacterial Agents; Kinetics; Article - Refereed
    • الرقم المعرف:
      10.1021.acs.biochem.2c00493
    • Note:
      English
    • Other Numbers:
      VPI oai:vtechworks.lib.vt.edu:10919/113361
      doi:10.1021/acs.biochem.2c00493
      EISSN:1520-4995
      ISSN:0006-2960
      1389867218
    • Contributing Source:
      VIRGINIA TECH
      From OAIster®, provided by the OCLC Cooperative.
    • الرقم المعرف:
      edsoai.on1389867218
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