In vitro reconstitution of indolmycin biosynthesis reveals the molecular basis of oxazolinone assembly

Abstract

The bacterial tryptophanyl-tRNA synthetase inhibitor indolmycin features a unique oxazolinone heterocycle whose biogenetic origins have remained obscure for over 50 years. Here we identify and characterize the indolmycin biosynthetic pathway, using systematic in vivo gene inactivation, in vitro biochemical assays, and total enzymatic synthesis. Our work reveals that a phenylacetateCoA ligase-like enzyme Ind3 catalyzes an unusual ATP-dependent condensation of indolmycenic acid and dehydroarginine, driving oxazolinone ring assembly. We find that Ind6, which also has chaperone-like properties, acts as a gatekeeper to direct the outcome of this reaction. With Ind6 present, the normal pathway ensues. Without Ind6, the pathway derails to an unusual shunt product. Our work reveals the complete pathway for indolmycin formation and sets the stage for using genetic and chemoenzymatic methods to generate indolmycin derivatives as potential therapeutic agents.

A network-based data-mining approach to investigate indole-related microbiota-host co-metabolism

“…The indole synthesis network was further filtered, by excluding reactions which are not undertaken by human or prokaryotic organisms. Three reactions ("rn:R00681", "rn:R01971", "rn:R01973") were included in the network despite not being linked to a KO, because there is evidence that these reactions can occur in some bacterial species (Jean and DeMoss 1968, Hornemann, Hurley et al 1971, Speedie, Hornemann et al 1975, Roberts and Rosenfeld 1977, Takai, Ushiro et al 1977, Du, Alkhalaf et al 2015.…”

Asymmetric C‐Alkylation by the S‐Adenosylmethionine‐Dependent Methyltransferase SgvM

“…Moreover,t he stereocenter formed can be prone to racemization, as in the case of 3-methylindolepyruvate or 3-methylphenylpyruvate.I nb iosynthesis,t his characteristic is exploited by subsequent enzymatic transamination and ketoreduction, resulting in the formation of vicinal stereocenters. [14,16] In most cases,t he absolute configuration of the stereocenter formed in the methylation step is not known. Furthermore,e nzymes catalyzing subsequent reaction steps could also influence the initially formed stereocenter.…”

Characterization of the Aminotransferase ThdN from Thienodolin Biosynthesis in Streptomyces albogriseolus

“…Milbredt ces sp. [8,9] Sequencea lignment with someo ther aminotransferases (Figure S1 in the Supporting Information) showedt hat the amino acids important for the formation of the tertiarya nd the quaternary structure of the protein (determined by protein BLAST against conservedd omains) are highly conserved among all these proteins. [2] The next-highest homology was with the tryptophan aminotransferase Ind8, identified to catalyzet he conversion of l-tryptophan into indole-3-pyruvate during indolmycin biosynthesis in Streptomycesg riseus subsp.…”

“…griseus. [8,9] Sequencea lignment with someo ther aminotransferases (Figure S1 in the Supporting Information) showedt hat the amino acids important for the formation of the tertiarya nd the quaternary structure of the protein (determined by protein BLAST against conservedd omains) are highly conserved among all these proteins. Thus, the PLP-binding site of ThdN might be formed by G115, S116, G117, F141, N189,Y 219, T245, S247, K248, and R256, with K248 as the catalytic residue, whereas G118, S151, V212, G254, L255, R256, and G283 seem to constitute the homodimer interface.…”