Role of Ser-257 in the Sliding Mechanism of NADP(H) in the Reaction Catalyzed by the Aspergillus fumigatus Flavin-dependent Ornithine N5-Monooxygenase SidA

Shirey, Carolyn M.; Badieyan, Somayesadat; Sobrado, Pablo

doi:10.1074/jbc.m113.487181

Cited by 23 publications

(32 citation statements)

References 36 publications

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“…For wtSidA a D k cat value of 3.70 ± 0.10 and a D ( k cat / K m ) value of 4.90 ± 0.50 were determined. These values are consistent with our previous work where it was determined that hydride transfer is partially rate limiting in wtSidA and that the reaction involves the stereospecific transfer of the 4- pro - R -4 2 H-of NADPH [9, 12]. With N323A, a D k cat value of 2.80 ± 0.10 and a D ( k cat / K m ) value of 2.70 ± 0.10 were determined.…”

Section: Resultssupporting

confidence: 91%

“…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%

“…1), also leads to an increase in the rate constant of flavin reduction. Molecular dynamics simulation showed that mutating Ser257 leads to increased motion of the nicotinamide ring permitting sampling of shorter distances between the NADPH- C4 and the N5-FAD [12]. We propose that in the case of N323A, deletion of the hydrogen bond with the ribose 3’-OH similarly leads to higher degrees of freedom of the nicotinamide ring during the sliding process, which increases the probability of hydride transfer.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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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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“…Furthermore, SidA and PvdA form long-lived stable C4a-hydroperoxyflavin intermediates which are the key oxygenating species responsible for substrate hydroxylation [21]. The mechanism of stabilization has been studied in detail in SidA and involves interactions of C4a-hydroperoxyflavin with NADP + , which remains bound throughout the catalytic cycle [15][16][17][18]22]. In contrast, NbtG does not form a stable C4ahydroperoxyflavin species, resulting in low levels of L-Lys hydroxylation (~30%) and high levels of superoxide and hydrogen peroxide by-products.…”

Section: Introductionmentioning

confidence: 99%

“…Sequence alignment shows that the Q/N residue is not conserved in NbtG and that a glutamate (Glu216) is 5 found instead. In PvdA and SidA, the Q/N residues (Gln217 and Gln256, respectively) have been proposed to be involved in NADP(H) binding; however, they have never been kinetically characterized [15,22,26]. In PvdA, Q217 is predicted to stabilize the loop conformation that exists between the first strand and the helix of the NADPH binding domain through hydrogen bonding with the backbone nitrogen of Asp289.…”

Section: Introductionmentioning

confidence: 99%

Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N-monooxygenase

Abdelwahab

Robinson

Rodrı́guez

et al. 2016

Archives of Biochemistry and Biophysics

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l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

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Flavin oxidation in flavin‐dependent N‐monooxygenases

et al. 2018

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Siderophore A (SidA) from Aspergillus fumigatus is a flavin-containing monooxygenase that hydroxylates ornithine (Orn) at the amino group of the side chain. Lysine (Lys) also binds to the active site of SidA; however, hydroxylation is not efficient and H O is the main product. The effect of pH on steady-state kinetic parameters was measured and the results were consistent with Orn binding with the side chain amino group in the neutral form. From the pH dependence on flavin oxidation in the absence of Orn, a pK value >9 was determined and assigned to the FAD-N5 atom. In the presence of Orn, the pH dependence displayed a pK value of 6.7 ±0.1 and of 7.70 ±0.10 in the presence of Lys. Q102 interacts with NADPH and, upon mutation to alanine, leads to destabilization of the C4a-hydroperoxyflavin (FAD ). Flavin oxidation with Q102A showed a pK value of ~8.0. The data are consistent with the pK of the FAD N5-atom being modulated to a value >9 in the absence of Orn, which aids in the stabilization of FAD . Changes in the FAD-N5 environment lead to a decrease in the pK value, which facilitates elimination of H O or H O. These findings are supported by solvent kinetic isotope effect experiments, which show that proton transfer from the FAD N5-atom is rate limiting in the absence of a substrate, however, is significantly less rate limiting in the presence of Orn and or Lys.

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Role of Ser-257 in the Sliding Mechanism of NADP(H) in the Reaction Catalyzed by the Aspergillus fumigatus Flavin-dependent Ornithine N5-Monooxygenase SidA

Cited by 23 publications

References 36 publications

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

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

Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N-monooxygenase

Flavin oxidation in flavin‐dependent N‐monooxygenases

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