A Tryptophan 6‐Halogenase and an Amidotransferase Are Involved in Thienodolin Biosynthesis

Abstract

The biosynthetic gene cluster for the plant growth-regulating compound thienodolin was identified in and cloned from the producer organism Streptomyces albogriseolus MJ286-76F7. Sequence analysis of a 27 kb DNA region revealed the presence of 21 ORFs, 14 of which are involved in thienodolin biosynthesis. Three insertional inactivation mutants were generated in the sequenced region to analyze their involvement in thienodolin biosynthesis and to functionally characterize specific genes. The gene inactivation experiments together with enzyme assays with enzymes obtained by heterologous expression and feeding studies showed that the first step in thienodolin biosynthesis is catalyzed by a tryptophan 6-halogenase and that the last step is the formation of a carboxylic amide group catalyzed by an amidotransferase. The results led to a hypothetical model for thienodolin biosynthesis.

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

“…In RebH and PrnA, L-tryptophan binds in the active site such that the C7–H bond is positioned proximal to the "Cl + " species and undergoes electrophilic aromatic substitution (41, 42). In addition to 7-Trp-FDHs (45, 46), both 5- (47–49) and 6-Trp-FDHs (50–54) have been characterized. As previously noted, site-selectivity for a single benzo-position, especially in the presence of the more electronically activated pyrrolo position, is remarkable.…”

Specific Enzymatic Halogenation—From the Discovery of Halogenated Enzymes to Their Applications In Vitro and In Vivo

“…[10,16,18,40,42,45] If the halogenase is involved in nonribosomal peptide or polyketide biosynthesis,i ti se xtremely unlikely that the halogenase substrate is af ree molecule.T his would only be the case if halogenation were the first step before activation of the amino acid for peptide synthesis or the starter unit for polyketide synthesis.T hus,i ti sn ot surprising that in abiosynthetic pathway,such as the one for welwitindolinone, which does not involve any peptidyl or acyl carrier proteins, [65] halogenation occurs at af ree substrate.1 2-epi-Fischerindole U( Scheme 11) and 12-epi-hapalindole Ca re chlorinated by the non-heme-iron halogenase WelO5. [10,16,18,40,42,45] If the halogenase is involved in nonribosomal peptide or polyketide biosynthesis,i ti se xtremely unlikely that the halogenase substrate is af ree molecule.T his would only be the case if halogenation were the first step before activation of the amino acid for peptide synthesis or the starter unit for polyketide synthesis.T hus,i ti sn ot surprising that in abiosynthetic pathway,such as the one for welwitindolinone, which does not involve any peptidyl or acyl carrier proteins, [65] halogenation occurs at af ree substrate.1 2-epi-Fischerindole U( Scheme 11) and 12-epi-hapalindole Ca re chlorinated by the non-heme-iron halogenase WelO5.…”

Characterization of the Aminotransferase ThdN from Thienodolin Biosynthesis in Streptomyces albogriseolus

“…For the conversion of 6-chlorotryptophan into 6-chlorothieno- [2,3-b]indole-2-carboxylic acid, the incorporation of as ulfur atom followed by ring formation/closure and oxidation building the thienyl ring is necessary.A ctivation of the a-carbono f6 -chlorotryptophan, catalyzed by an aminotransferase to form 6-chloroindole-3-pyruvate (Scheme 1), was suggestedt of acilitate sulfur incorporation. [6] This was confirmedb yW ang et al, who recently described the isolation of the thienodolin biosynthetic gene cluster from Streptomyces sp.F XJ1.172a nd showed the in vitro aminotransferase activity of ThnJ (homologue of ThdN). [6] This was confirmedb yW ang et al, who recently described the isolation of the thienodolin biosynthetic gene cluster from Streptomyces sp.F XJ1.172a nd showed the in vitro aminotransferase activity of ThnJ (homologue of ThdN).…”