A [6+4]-cycloaddition adduct is the biosynthetic intermediate in streptoseomycin biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Biosynthesis

Background:

• Streptoseomycin (STM) is a bacterial macrolactone with a complex pentacyclic ring structure.
• Previous work identified the STM biosynthetic gene cluster and the StmD bispericyclase, forming 6/10/6- and 10/6/6-tricyclic adducts.
• The mechanism for installing and stabilizing the 10/6/6-tricyclic core remained unelucidated.

Purpose of the Study:

• To identify the enzymes responsible for forming the stable 10/6/6-tricyclic core of Streptoseomycin.
• To elucidate the specific steps and intermediates involved in the late-stage biosynthesis of Streptoseomycin.
• To determine the bona fide biosynthetic intermediate leading to the 10/6/6-tricyclic core.

Main Methods:

• Identification and characterization of oxidoreductases involved in Streptoseomycin biosynthesis.
• Enzymatic assays using StmO1, StmO2, and StmK.
• Analysis of reaction intermediates, including spontaneous rearrangements and tautomerization.
• Crystal structure determination of NtfO1-NtfO2 complex, homologues of StmO1-StmO2.

Main Results:

• Three oxidoreductases, StmO1, StmO2, and StmK, were identified as crucial for stabilizing the 10/6/6-tricyclic core.
• Flavin-dependent oxidoreductases StmO1 and StmO2 catalyze hydroxylation of the [6+4]-adduct.
• A spontaneous [3,3]-Cope rearrangement and enol-ketone tautomerization lead to a 10/6/6-tricyclic intermediate, which StmK reduces to a stable alcohol.
• Crystal structure of NtfO1-NtfO2 revealed protein-protein interactions, suggesting a functional complex.

Conclusions:

• The [6+4]-adduct, not the [4+2]-adduct, is the true biosynthetic intermediate for the 10/6/6-tricyclic core.
• StmO1, StmO2, and StmK collectively establish the 10/6/6-tricyclic core through hydroxylation and ketoreduction.
• The findings provide key insights into the late-stage enzymatic steps in Streptoseomycin biosynthesis.