Lysine:N6-Hydroxylase: Cofactor Interactions

Marrone, Laura; Beecroft, M.; Viswanatha, T.

doi:10.1006/bioo.1996.0027

Cited by 9 publications

(6 citation statements)

References 24 publications

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“…This is not the first time that ''oxidative addition,'' effectively equivalent to substitution, has been observed in reactions between thiols and quinonimines or quinones of relevance to biochemical studies. A similar pathway has been shown to operate with 2,6-dichloroindophenol, a widely used redox indicator (13)(14)(15). Recently, oxidative addition of cysteine to the 1,2-benzoquinone formed by oxidation of epinephrine has been extensively studied (16).…”

Section: Discussionmentioning

confidence: 93%

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

Kitson

1998

Bioorganic Chemistry

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Section: Discussionmentioning

confidence: 93%

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

Kitson

1998

Bioorganic Chemistry

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“…Hydrogen peroxide was assayed according to an established procedure (10). Hydrogen peroxide in the assay mixture was calculated from absorbance at 480 nm and an extinction coefficient of 3,500 M Ϫ1 cm Ϫ1 , determined with known concentrations of standard hydrogen peroxide.…”

Section: Methodsmentioning

confidence: 99%

Heterologous Expression, Purification, and Characterization of an l -Ornithine N ⁵ -Hydroxylase Involved in Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa

Seah

2006

J Bacteriol

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Pseudomonas aeruginosa is an opportunistic pathogen that produces the siderophore pyoverdine, which enables it to acquire the essential nutrient iron from its host. Formation of the iron-chelating hydroxamate functional group in pyoverdine requires the enzyme PvdA, a flavin-dependent monooxygenase that catalyzes the N 5 hydroxylation of L-ornithine. pvdA from P. aeruginosa was successfully overexpressed in Escherichia coli, and the enzyme was purified for the first time. The enzyme possessed its maximum activity at pH 8.0. In the absence of L-ornithine, PvdA has an NADPH oxidase activity of 0.24 ؎ 0.02 mol min ؊1 mg ؊1 . The substrate L-ornithine stimulated this activity by a factor of 5, and the reaction was tightly coupled to the formation of hydroxylamine. The enzyme is specific for NADPH and flavin adenine dinucleotide (FAD ؉ ) as cofactors, as it cannot utilize NADH and flavin mononucleotide. By fluorescence titration, the dissociation constants for NADPH and FAD ؉ were determined to be 105.6 ؎ 6.0 M and 9.9 ؎ 0.3 M, respectively. Steady-state kinetic analysis showed that the L-ornithine-dependent NADPH oxidation obeyed Michaelis-Menten kinetics with apparent K m and V max values of 0.58 mM and 1.34 mol min ؊1 mg ؊1 . L-Lysine was a nonsubstrate effector that stimulated NADPH oxidation, but uncoupling occurred and hydrogen peroxide instead of hydroxylated L-lysine was produced. L-2,4-Diaminobutyrate, L-homoserine, and 5-aminopentanoic acid were not substrates or effectors, but they were competitive inhibitors of the L-ornithine-dependent NADPH oxidation reaction, with K ic s of 3 to 8 mM. The results indicate that the chemical nature of effectors is important for simulation of the NADPH oxidation rate in PvdA.Iron is an essential nutrient for most organisms. In humans, iron is sequestered by iron binding proteins such as transferrin, lactoferrin, and hemoglobin and is therefore unavailable to invading microbial pathogens. Microorganisms can counter this nonspecific host defense mechanism by the production of low-molecular-weight, high iron(III) affinity molecules termed siderophores (4, 25). Siderophores are synthesized intracellularly and then secreted into the environment, where they chelate and deliver the complexed iron back into the cell via specific membrane transporters. Most siderophores are peptides, and the functional groups that chelate iron(III) include catecholates, carboxylates, and hydroxamates (15).The hydroxamate functional groups are usually derived from diamino acids such as L-lysine and L-ornithine. A flavin-dependent monooxygenase (EC 1.14.13) catalyzes the first step in the formation of hydroxamates by oxidizing the terminal amino group of the respective substrates to produce the corresponding hydroxylamines (Fig. 1). On the basis of studies with the Escherichia coli L-lysine N 6 -hydroxylase (IucD) and by analogy to other flavin monooxygenases (1), this reaction is proposed to proceed in two steps. In the reductive half reaction, flavin adenine dinucleotide (FAD ϩ ) is reduced by NADP...

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“…Similar stringency in substrate preference appears to be common in FMOs involved in siderophore biosynthesis. 12–14,21–23 We have therefore sought to understand how Af-OMO achieves such specificity in spite of its bold mechanism. Notably, no crystal structure for a substrate-stringent FMO yet exists (see refs 24 and 25).…”

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confidence: 99%

Regulated O₂ Activation in Flavin-Dependent Monooxygenases

2011

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Flavin-dependent monooxygenases (FMOs) are involved in important biosynthetic pathways in diverse organisms, including production of the siderophores used for the import and storage of essential iron in serious pathogens. We have shown that the FMO from Aspergillus fumigatus, an ornithine monooxygenase (Af-OMO), is mechanistically similar to its well-studied distant homologues from mammalian liver. The latter are highly promiscuous in their choice of substrates, while Af-OMO is unusually specific. This presents a puzzle: how do Af-OMO and other FMOs of the biosynthetic classes achieve such specificity? We have discovered substantial enhancement in the rate of O2 activation in Af-OMO in the presence of l-arginine, which acts as a small molecule regulator. Such protein-level regulation could help explain how this and related biosynthetic FMOs manage to couple O2 activation and substrate hydroxylation to each other and to the appropriate cellular conditions. Given the essentiality of Fe to Af and the avirulence of the Af-OMO gene knock out, inhibitors of Af-OMO are likely to be drug targets against this medically intractable pathogen.

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Lysine:N6-Hydroxylase: Cofactor Interactions

Cited by 9 publications

References 24 publications

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

Heterologous Expression, Purification, and Characterization of an l -Ornithine N ⁵ -Hydroxylase Involved in Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa

Regulated O₂ Activation in Flavin-Dependent Monooxygenases

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Lysine:N6-Hydroxylase: Cofactor Interactions

Cited by 9 publications

References 24 publications

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

Heterologous Expression, Purification, and Characterization of an l -Ornithine N 5 -Hydroxylase Involved in Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa

Regulated O2 Activation in Flavin-Dependent Monooxygenases

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Heterologous Expression, Purification, and Characterization of an l -Ornithine N ⁵ -Hydroxylase Involved in Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa

Regulated O₂ Activation in Flavin-Dependent Monooxygenases