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Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis.

ABSTRACT: N-Hydroxylating monooxygenases (NMOs) are involved in siderophore biosynthesis. Siderophores are high affinity iron chelators composed of catechol and hydroxamate functional groups that are synthesized and secreted by microorganisms and plants. Recently, a new siderophore named albachelin was isolated from a culture of Amycolatopsis alba growing under iron-limiting conditions. This work focuses on the expression, purification, and characterization of the NMO, abachelin monooxygenase (AMO) from A. alba. This enzyme was purified and characterized in its holo (FAD-bound) and apo (FAD-free) forms. The apo-AMO could be reconstituted by addition of free FAD. The two forms of AMO hydroxylate ornithine, while lysine increases oxidase activity but is not hydroxylated and display low affinity for NADPH.

SUBMITTER: Bufkin K

PROVIDER: S-EPMC6151521 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis.

Bufkin Kendra K Sobrado Pablo P

Molecules (Basel, Switzerland) 20171001 10

<i>N</i>-Hydroxylating monooxygenases (NMOs) are involved in siderophore biosynthesis. Siderophores are high affinity iron chelators composed of catechol and hydroxamate functional groups that are synthesized and secreted by microorganisms and plants. Recently, a new siderophore named albachelin was isolated from a culture of <i>Amycolatopsis alba</i> growing under iron-limiting conditions. This work focuses on the expression, purification, and characterization of the NMO, abachelin monooxygenas ...[more]

PMID: 28974024

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Project description:Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa. I117 contained an insertion in a pvdA homolog, the gene for the enzyme L-ornithine N(5)-oxygenase, which catalyzes the hydroxylation of L-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursor L-N(5)-OH-Orn was added to the culture medium. T10 contained an insertion in a pvdD homolog, which is a peptide synthetase involved in pyoverdine synthesis. beta-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn5-OT182 contains a lacZ gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The B. cepacia pvdA homolog was approximately 47% identical and 59% similar to L-ornithine N(5)-oxygenase from P. aeruginosa. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of (59)Fe-ornibactin uptake in I117. A chromosomal pvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. The pvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functional pvdA gene appears to be required for effective colonization and persistence in B. cepacia lung infections.

Dataset Information

Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis.

Publications

Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis.

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