Activation and Loading of the Starter Unit during Thiocoraline Biosynthesis.

The initiation of the nonribosomal peptide synthetase (NRPS) assembly of the bisintercalator natural product thiocoraline involves key enzymatic steps for AMP activation and carrier protein loading of the starter unit 3-hydroxyquinaldic acid (3HQA). Gene cluster data combined with protein sequence homology analysis originally led us to propose that TioJ could be responsible for the AMP activation step, whereas TioO could act as the thiolation (T) domain, facilitating the transfer of 3HQA to the next NRPS module, TioR. Herein, we confirmed the involvement of TioJ in thiocoraline biosynthesis by tioJ knockout and in vitro activation of 3HQA studies.

Enzymatic Evidence for a Revised Congocidine Biosynthetic Pathway.

Naturally produced pyrrolamides, such as congocidine, are nonribosomal peptides that bind to the minor groove of DNA. Efforts to delineate the biosynthetic machinery responsible for their assembly have mainly employed genetic methods, and the enzymes responsible for their biosynthesis remain largely uncharacterized. We report the biochemical characterization of four proteins involved in congocidine formation: the adenylation-thiolation (A-T) di-domain Cgc18(1-610), its MbtH-like partner SAMR0548, the AMP-binding enzyme Cgc3*, and the T domain Cgc19.

Adenylation and S-methylation of cysteine by the bifunctional enzyme TioN in thiocoraline biosynthesis.

The antitumor agent thiocoraline is a nonribosomally biosynthesized bisintercalator natural product, which contains in its peptidic backbone two S-methylated l-cysteine residues. S-Methylation occurs very rarely in nature, and is observed extremely rarely in nonribosomal peptide scaffolds. We have proposed that during thiocoraline biosynthesis, TioN, a stand-alone adenylation domain interrupted by the S-adenosyl-l-methionine binding region of a methyltransferase enzyme, is capable of performing two functions: the adenylation and S-methylation of l-cysteine.

Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases.

The lipoamino acids and endovanilloids have multiple roles in nociception, pain, and inflammation, yet their biological reactivity has not been fully characterized. Cyclooxygenases (COXs) and lipoxygenases (LOs) oxygenate polyunsaturated fatty acids to generate signaling molecules. The ability of COXs and LOs to oxygenate arachidonyl-derived lipoamino acids and vanilloids was investigated.