Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions

Herrero, Marta; Lorenzo, Vı́ctor de; Neilands, J. B.

doi:10.1128/jb.170.1.56-64.1988

Cited by 73 publications

(55 citation statements)

References 53 publications

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“…These codons can be read as tryptophan (W) in a supK strain (28). Shaded bases in the iucD sequence indicate the Shine-Dalgarno sequence as reported by Herrero et al (17). IucD (Fig.…”

Section: Resultsmentioning

confidence: 91%

“…In a previous study, Herrero et al (17) suggested that IucD is an integral membrane protein associated with the inner membrane by at least two attachment sites. One of the membrane attachment sites appears to consist of a hydrophobic amino acid sequence resembling a leader peptide located on the amino terminus of IucD (17). The features in primary sequences of proteins determining membrane insertion are not completely elucidated (34).…”

Section: Resultsmentioning

confidence: 99%

“…The DNA sequences of the fusion end points in pAT2, pAT3, and pATS were deduced from the published sequences of pUC19 and iucD (17,37). Shaded amino acids indicate the portion of the recombinant polypeptide contributed by the 0-galactosidase a-peptide encoded by pUC19.…”

Section: Resultsmentioning

confidence: 99%

“…The inability to obtain a soluble form of IucD should not be unexpected in view of the presence of domains capable of association with the inner membrane (17). The present study concerns a genetic strategy to construct soluble forms of recombinant IucD proteins with altered amino termini by creating a series of in-frame gene (15).…”

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confidence: 99%

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Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

et al. 1993

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The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:NM-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme 13-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of 1-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, lucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state. The enzyme was stringently specific with regard to its substrate and cofactor, L-lysine and flavin adenine dinucleotide, respectively. NADPH appeared to be the preferred electron donor used by IucD439 in the N hydroxylation process.

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“…These codons can be read as tryptophan (W) in a supK strain (28). Shaded bases in the iucD sequence indicate the Shine-Dalgarno sequence as reported by Herrero et al (17). IucD (Fig.…”

Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

et al. 1993

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“…Both enzymes hydroxylate the primary amines of amino acid sidechains (Figure 1), and share 46 % sequence similarity. The structure of IucD has not been determined and there has been debate about the solubility and cellular localization (membrane or cytoplasm) of this enzyme (25)(26)(27)(28). Initial attempts to overexpress IucD led to insoluble protein, and hydrophobic stretches of the enzyme were identified that could serve as transmembrane helices (27).…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of a Flavin Adenine Dinculeotide-Dependent Monooxygenase, Ornithine Hydroxylase fromPseudomonas aeruginosa, Suggests a Novel Reaction Mechanism

Meneely

Lamb

2007

Biochemistry

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Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both FAD and NADPH for activity, and was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics using an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a non-substrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA in relation to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (para-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady state experiments indicate that PvdA:FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to para-hydroxybenzoate hydroxylase (PHBH from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism. P. aeruginosa is an opportunistic human pathogen that under iron-limiting conditions produces two siderophores, pyochelin and pyoverdin, which contribute to virulence (1,2). Pyoverdin, a hydroxamate siderophore, chelates iron with high affinity, and has the ability to remove iron from host proteins such as transferrin and lactoferrin (3). Derivatives of ornithine, both hydroxyornithine and formyl-hydroxyornithine, are incorporated into the peptide backbone of pyoverdin and directly coordinate the iron (4).Ornithine hydroxylase (PvdA or L-ornithine N 5 -oxygenase) is the first enzyme involved in the derivatization of the ornithine, hydroxylating the primary amine of the side chain ( Figure 1a). PvdA is part of the pyoverdin locus (5) and PvdA deletion mutants are pyoverdin-deficient (6, 7). PvdA is functionally related to the lysine hydroxylase (IucD) of E. coli, which is required for production of the aerobactin siderophore (Figure 1b), para-hydroxybenzoate hydroxylase (PHBH) of the soil bacterium P. fluorescens, which is important in the biodegradation of lignin from wood (Figure 1c), and flavin-containing monooxygenases (FMOs) from a variety of organisms from bacteria to mammals involved in xenobiotic detoxification (Figure 1d) . FAD is reduced by NADPH and can then donate electrons to molecular oxygen ( Figure 1). The end result is the production of H 2 O, and a hydroxyl group is added to the sidechain amine of lysine (IucD), the activated aromatic ring of the hydroxybenzoate (PHBH), or to a ...

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Lysine:N6-Hydroxylase: Cofactor Interactions

Marrone¹,

Beecroft²,

Viswanatha³

1996

Bioorganic Chemistry

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Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions

Cited by 73 publications

References 53 publications

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

Biochemical Characterization of a Flavin Adenine Dinculeotide-Dependent Monooxygenase, Ornithine Hydroxylase fromPseudomonas aeruginosa, Suggests a Novel Reaction Mechanism

Lysine:N6-Hydroxylase: Cofactor Interactions

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