Structure and function of a flavin-dependent S-monooxygenase from garlic (Allium sativum)

Valentino, Hannah; Campbell, A.C.; Schuermann, Jonathan P.; Sultana, Nazneen; Nam, Han G.; Leblanc, Sophie; Tanner, John J.; Sobrado, Pablo

doi:10.1074/jbc.ra120.014484

Cited by 19 publications

(11 citation statements)

References 66 publications

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“…5 ), proving the prerequisite of the oxepin ring for an acceptance by OpaC. The kinetic data of the OpaC reaction with 4 in the presence of NADPH fit well to a typical velocity equation with substrate inhibition 31 , 32 . An apparent K m value at 0.43 ± 0.04 mM, a turnover number ( k cat ) at 0.16 ± 0.01 s −1 , the catalytic efficiency ( k cat / K m ) at 0.37 mM −1 s −1 and a substrate inhibition constant ( K i ) at 0.39 ± 0.03 mM (Fig.…”

Section: Resultssupporting

confidence: 53%

Oxepinamide F biosynthesis involves enzymatic d-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration

et al. 2020

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Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. Here we report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. The nonribosomal peptide synthase (NRPS) OpaA assembles the quinazolinone core with d-Phe incorporation. The cytochrome P450 enzyme OpaB catalyzes alone the oxepin ring formation. The flavoenzyme OpaC installs subsequently one hydroxyl group at the oxepin ring, accompanied by double bond migration. The epimerase OpaE changes the d-Phe residue back to l-form, which is essential for the final methylation by OpaF.

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Section: Resultssupporting

confidence: 53%

Oxepinamide F biosynthesis involves enzymatic d-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration

et al. 2020

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“…The presence of two phases for flavin reduction has been previously observed in some Class B FMOs. 22,53 The fast phase, occurring within the first second, is faster than the k cat measured in the steady-state kinetic assays. The slow phase, making up approximately 25% of the amplitude change for the reduction, occurs between the first and tenth seconds of reduction.…”

Section: ■ Discussionmentioning

confidence: 98%

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

et al. 2022

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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+.

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“…The following references are cited in the supporting information for this article: Franceschini et al (2012) and Valentino et al (2020).…”

Section: Related Literaturementioning

confidence: 99%

Structural basis of regioselective tryptophan dibromination by the single-component flavin-dependent halogenase AetF

Gäfe¹,

Niemann²

2023

Acta Crystallogr D Struct Biol

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The flavin-dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7-dibromotryptophan. In contrast to the well studied two-component tryptophan halogenases, AetF is a single-component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single-component FDH. Rotational pseudosymmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin-dependent monooxygenases. It contains two dinucleotide-binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Å apart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5-bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5-bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7-bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single-component FDHs.

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Structure and function of a flavin-dependent S-monooxygenase from garlic (Allium sativum)

Cited by 19 publications

References 66 publications

Oxepinamide F biosynthesis involves enzymatic d-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration

Oxepinamide F biosynthesis involves enzymatic d-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration

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

Structural basis of regioselective tryptophan dibromination by the single-component flavin-dependent halogenase AetF

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