An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

Binda, Claudia; Robinson, Reeder M.; Campo, Julia S. Martín del; Keul, Nicholas D.; Rodrı́guez, P.; Robinson, Howard; Mattevi, Andrea; Sobrado, Pablo

doi:10.1074/jbc.m114.629485

Cited by 41 publications

(59 citation statements)

References 45 publications

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“…S6A-B), which are completely consistent with the fragmentation pattern of the positive control (the NbtG reaction) (Fig. S6C-D, Table S4) (22). However, the reaction (without enzyme added as negative control) mixtures, as detected by LC-MS, could not produce the expected MS peaks (Fig.…”

Section: Resultssupporting

confidence: 83%

Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction ofC-nucleoside scaffolds

Zhang

et al. 2019

Preprint

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24Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside 25 antibiotics, in which ribose and a pyrazole-derived base are linked by a C-glycosidic 26 bond, however, the logic underlying the biosynthesis of these molecules has 27 remained largely unexplored. Here, we report the discovery of the pathways for 28 FOR-A and PRF-A biosynthesis from diverse actinobacteria, and demonstrate that 29 their biosynthesis is initiated by a lysine N 6 -monooxygenase. Moreover, we show 30 that the forT and prfE (individually related to FOR-A and PRF-A biosynthesis) 31 mutants are correspondingly capable of accumulating the unexpected 32 pyrazole-related intermediates, compound 11 and 9a. We also decipher the 33 enzymatic basis of ForT/PrfE for the C-glycosidic bond formation in FOR-A/PRF-A 34 biosynthesis. To our knowledge, ForT/PrfE represents the first example of β-RFA-P 35 (β-ribofuranosyl-aminobenzene 5'-phosphate) synthase-like enzymes governing 36 C-nucleoside scaffold construction in natural product biosynthesis. These data 37 establish a foundation for combinatorial biosynthesis of related purine nucleoside 38 antibiotics, and also open the way for target-directed genome mining of 39

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Section: Resultssupporting

confidence: 83%

Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction ofC-nucleoside scaffolds

Zhang

et al. 2019

Preprint

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“…It has been proposed for wtSidA and other Class B flavin monoxygenases that domain rotation occurs to allow NADP(H) to “slide” into the conformation required for FAD OOH formation after hydride transfer [12, 16, 44, 45]. It is possible that Asn323 is involved in guiding the NR group during the reduction before it acquires the position required for FAD OOH stabilization (Fig.…”

Section: Discussionmentioning

confidence: 99%

Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase

Robinson

Qureshi

Klancher

et al. 2015

Archives of Biochemistry and Biophysics

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The SidA ornithine N5-monooxygenase from Aspergillus fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP(+) and ornithine shows that the nicontinamide riboside group of NADP is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites.

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“…52 Unlike the canonical Class B NMOs, NbtG releases unusually high amounts of uncoupled hydrogen peroxide and superoxide. While all the key residues for binding ornithine in SidA are conserved in NbtG, its NADPH binding domain is significantly rotated compared to that of SidA.…”

Section: N–o Bond Forming Enzymesmentioning

confidence: 99%

“…However, Sobrado et al have suggested that NbtsG may have evolved as a dual NADPH oxidase. 52 Peroxide production by this enzyme may also serve signaling roles in response to amino acid levels inside the cell. 76 …”

Section: N–o Bond Forming Enzymesmentioning

confidence: 99%

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

et al. 2017

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Natural products that contain functional groups with heteroatom-heteroatom linkages (X–X, where X = N, O, S, and P) are a small yet intriguing group of metabolites. The reactivity and diversity of these structural motifs has captured the interest of synthetic and biological chemists alike. Functional groups containing X–X bonds are found in all major classes of natural products and often impart significant biological activity. This review presents our current understanding of the biosynthetic logic and enzymatic chemistry involved in the construction of X–X bond containing functional groups within natural products. Elucidating and characterizing biosynthetic pathways that generate X–X bonds could both provide tools for biocatalysis and synthetic biology, as well as guide efforts to uncover new natural products containing these structural features.

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An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

Cited by 41 publications

References 45 publications

Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction ofC-nucleoside scaffolds

Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction ofC-nucleoside scaffolds

Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

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