Benzastatins have unique structures probably derived from geranylated p-aminobenzoic acids. The indoline and tetrahydroquinoline scaffolds are presumably formed by cyclization of the geranyl moiety, but the cyclization mechanism was unknown. We studied the benzastatin biosynthetic gene cluster of Streptomyces sp. RI18; functions of the six enzymes encoded by it were analyzed by gene disruption in a heterologous host and in vitro enzyme assays. We propose the biosynthetic pathway for benzastatins in which a cytochrome P450 (BezE) is responsible for the cyclization of geranylated p-acetoxyaminobenzoic acids; BezE catalyzes elimination of acetic acid to form an iron nitrenoid, nitrene transfer to form an aziridine ring, and nucleophilic addition of hydroxide ion to C-10 and chloride ion to C-9 to generate the indoline and tetrahydroquinoline scaffolds, respectively. Discovery of this enzyme, which should be termed cytochrome P450 nitrene transferase, provides an important insight into the functional diversity of cytochrome P450.
Prenyl pyrophosphate methyltransferases enhance the structural diversity of terpenoids.H owever,t he molecular basis of their catalytic mechanisms is poorly understood. In this study,u sing multiple strategies,w ec haracterized ag eranyl pyrophosphate (GPP) C6-methyltransferase,BezA. Biochemical analysis revealed that BezA requires Mg 2+ and solely methylates GPP.T he crystal structures of BezA and its complex with S-adenosyl homocysteine were solved at 2.10 and 2.56 ,r espectively.F urther analyses using site-directed mutagenesis,m olecular docking, molecular dynamics simulations,a nd quantum mechanics/molecular mechanics calculations revealed the molecular basis of the methylation reaction. Importantly,t he function of E170 as ac atalytic base to complete the methylation reaction was established. We also succeeded in switching the substrate specificity by introducing aW 210A substitution, resulting in an unprecedented farnesyl pyrophosphate C6-methyltransferase.
Prenyl pyrophosphate methyltransferases enhance the structural diversity of terpenoids.H owever,t he molecular basis of their catalytic mechanisms is poorly understood. In this study,u sing multiple strategies,w ec haracterized ag eranyl pyrophosphate (GPP) C6-methyltransferase,BezA. Biochemical analysis revealed that BezA requires Mg 2+ and solely methylates GPP.T he crystal structures of BezA and its complex with S-adenosyl homocysteine were solved at 2.10 and 2.56 ,r espectively.F urther analyses using site-directed mutagenesis,m olecular docking, molecular dynamics simulations,a nd quantum mechanics/molecular mechanics calculations revealed the molecular basis of the methylation reaction. Importantly,t he function of E170 as ac atalytic base to complete the methylation reaction was established. We also succeeded in switching the substrate specificity by introducing aW 210A substitution, resulting in an unprecedented farnesyl pyrophosphate C6-methyltransferase.
Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6‐5‐3 tricyclic system containing an iminium cation from Streptomyces griseus. The gene cluster has 22 genes, including four genes encoding polyketide synthases (PKSs), which consist of eight modules in total. In vitro analysis of the first module revealed that its acyltransferase domain selects malonyl‐CoA, although predicted to select methylmalonyl‐CoA. Inactivation of seven tailoring enzyme‐encoding genes and structural elucidation of four compounds accumulated in mutants provided important insights into iminimycin biosynthesis, although some of these compounds appeared to be shunt products. This study expands our knowledge of the biosynthetic machinery of indolizidine alkaloids and the enzymatic chemistry of PKS.
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