Studies with Lysine N6-Hydroxylase. Effect of a Mutation in the Assumed FAD Binding Site on Coenzyme Affinities and on Lysine Hydroxylating Activity

Stehr, Matthias; Smau, Liliana; Singh, Mahavir; Seth, Oliver; Macheroux, Peter; Ghisla, Sandro; Diekmann, H.

doi:10.1515/bc.1999.006

Cited by 15 publications

(17 citation statements)

References 23 publications

(47 reference statements)

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“…A bar showing 10% divergence is included. In addition to the monooxygenases that are discussed in the text some other annotated sequences are indicated (PNMO: putrescine hydroxylase from Bordetella bronchiseptica Q44740 [29]; DNMO: N 4 ‐diaminopropane monooxygenase from Sinorhizobium meliloti , AAK65920 [30]; ONMO1: L ‐ornithine N 5 ‐monooxygenase from Ustilago maydis , P56584 [31]; ONMO2: L ‐ornithine N 5 ‐monooxygenase from P. aeruginosa (Q51548) [32]; ONMO3: L ‐ornithine N 5 ‐monooxygenase from Pseudomonas strain B10 (AAG27518) [26]). The clustering of the three monooxygenase families is indicated by gray shading.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Baeyer–Villiger monooxygenase sequence motif

et al. 2002

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Baeyer^Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C^C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXHXXXW(P/D). Studies with site-directed mutants of 4-hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD-dependent monooxygenases: the so-called flavin-containing monooxygenases (FMOs), the N-hydroxylating monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank. ß

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Section: Resultsmentioning

confidence: 99%

Identification of a Baeyer–Villiger monooxygenase sequence motif

et al. 2002

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“…The type 1 signature of the FAD-dependent oxidoreductases (motif GXGXXG; residues 16 to 21 of PsbA) shows a typical replacement of the last glycine with proline; this is a unique feature of hydroxylases involved in siderophore biosynthesis (34). Although such deviation is predicted to distort the ␣-helical structure within the secondary ␤␣␤ fingerprint of flavin-binding proteins (45), recent studies suggest that the deviated sequence is still compatible with the binding of FAD and association with the cytoplasmic membrane (35,36). The existence of an additional dinucleotide (NADP)-binding site is inferred from the identification of a type 3 signature of the FAD-dependent oxidoreductases [GXGXX(G/A)] in the central portion of all the members of the group (residues 214 to 219 of PsbA) (34).…”

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confidence: 99%

Pseudobactin Biogenesis in the Plant Growth-Promoting Rhizobacterium Pseudomonas Strain B10: Identification and Functional Analysis of the l -Ornithine N ⁵ -Oxygenase ( psbA ) Gene

et al. 2000

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“…The procedure was similar to that reported for the assay of lysine N 6 -hydroxylase activity reported previously (19). The concentration of hydroxylamine produced by the enzyme was calculated from the extinction coefficient of 12,500 M Ϫ1 cm Ϫ1 determined from a standard curve of known concentrations of hydroxylamine hydrochloride.…”

Section: Methodsmentioning

confidence: 96%

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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Studies with Lysine N6-Hydroxylase. Effect of a Mutation in the Assumed FAD Binding Site on Coenzyme Affinities and on Lysine Hydroxylating Activity

Cited by 15 publications

References 23 publications

Identification of a Baeyer–Villiger monooxygenase sequence motif

Identification of a Baeyer–Villiger monooxygenase sequence motif

Pseudobactin Biogenesis in the Plant Growth-Promoting Rhizobacterium Pseudomonas Strain B10: Identification and Functional Analysis of the l -Ornithine N ⁵ -Oxygenase ( psbA ) Gene

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

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Studies with Lysine N6-Hydroxylase. Effect of a Mutation in the Assumed FAD Binding Site on Coenzyme Affinities and on Lysine Hydroxylating Activity

Cited by 15 publications

References 23 publications

Identification of a Baeyer–Villiger monooxygenase sequence motif

Identification of a Baeyer–Villiger monooxygenase sequence motif

Pseudobactin Biogenesis in the Plant Growth-Promoting Rhizobacterium Pseudomonas Strain B10: Identification and Functional Analysis of the l -Ornithine N 5 -Oxygenase ( psbA ) Gene

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

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Pseudobactin Biogenesis in the Plant Growth-Promoting Rhizobacterium Pseudomonas Strain B10: Identification and Functional Analysis of the l -Ornithine N ⁵ -Oxygenase ( psbA ) Gene

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