Dataset Information

In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis.

ABSTRACT: p-Aminobenzoate N-oxygenase (AurF) from Streptomyces thioluteus catalyzes the formation of unusual polyketide synthase starter unit p-nitrobenzoic acid (pNBA) from p-aminobenzoic acid (pABA) in the biosynthesis of antibiotic aureothin. AurF is a metalloenzyme, but its native enzymatic activity has not been demonstrated in vitro, and its catalytic mechanism is unclear. In addition, the nature of the cofactor remains a controversy. Here, we report the in vitro reconstitution of the AurF enzyme activity, the crystal structure of AurF in the oxidized state, and the cocrystal structure of AurF with its product pNBA. Our combined biochemical and structural analysis unequivocally indicates that AurF is a non-heme di-iron monooxygenase that catalyzes sequential oxidation of aminoarenes to nitroarenes via hydroxylamine and nitroso intermediates.

SUBMITTER: Choi YS

PROVIDER: S-EPMC2383931 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis.

Choi Yoo Seong YS Zhang Houjin H Brunzelle Joseph S JS Nair Satish K SK Zhao Huimin H

Proceedings of the National Academy of Sciences of the United States of America 20080505 19

p-Aminobenzoate N-oxygenase (AurF) from Streptomyces thioluteus catalyzes the formation of unusual polyketide synthase starter unit p-nitrobenzoic acid (pNBA) from p-aminobenzoic acid (pABA) in the biosynthesis of antibiotic aureothin. AurF is a metalloenzyme, but its native enzymatic activity has not been demonstrated in vitro, and its catalytic mechanism is unclear. In addition, the nature of the cofactor remains a controversy. Here, we report the in vitro reconstitution of the AurF enzyme act ...[more]

PMID: 18458342

Similar Datasets

Project description:Chlamydia trachomatis lacks the canonical genes required for the biosynthesis of p-aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from C. trachomatis, the CT610 gene, that rescues Escherichia coli ΔpabA, ΔpabB, and ΔpabC mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site. Genetic studies ruled out several potential substrates for CT610-dependent pABA biosynthesis, including chorismate and other shikimate pathway intermediates, leaving the actual precursor(s) unknown. Here, we supplied isotopically labeled potential precursors to E. coli ΔpabA cells expressing CT610 and found that the aromatic portion of tyrosine was highly incorporated into pABA, indicating that tyrosine is a precursor for CT610-dependent pABA biosynthesis. Additionally, in vitro enzymatic experiments revealed that purified CT610 exhibits low pABA synthesis activity under aerobic conditions in the absence of tyrosine or other potential substrates, where only the addition of a reducing agent such as dithiothreitol appears to stimulate pABA production. Furthermore, site-directed mutagenesis studies revealed that two conserved active site tyrosine residues are essential for the pABA synthesis reaction in vitro Thus, the current data are most consistent with CT610 being a unique self-sacrificing enzyme that utilizes its own active site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O2 and a reduced diiron cofactor.IMPORTANCEChlamydia trachomatis is the most reported sexually transmitted infection in the United States and the leading cause of infectious blindness worldwide. Unlike many other intracellular pathogens that have undergone reductive evolution, C. trachomatis is capable of de novo biosynthesis of the essential cofactor tetrahydrofolate using a noncanonical pathway. Here, we identify the biosynthetic precursor to the p-aminobenzoate (pABA) portion of folate in a process that requires the CT610 enzyme from C. trachomatis We further provide evidence that CT610 is a self-sacrificing or "suicide" enzyme that uses its own amino acid residue(s) as the substrate for pABA synthesis. This work provides the foundation for future investigation of this chlamydial pABA synthase, which could lead to new therapeutic strategies for C. trachomatis infections.

Dataset Information

In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis.

Publications

In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets