Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Organic Chemistry

Background:

• Diels-Alder-like [4+2] cyclizations are key in spirotetronate and spirotetramate antibiotic biosynthesis.
• The biosynthesis of the polyether antibiotic tetronasin was not expected to involve such cyclization steps.
• The tetronasin gene cluster contains Tsn11 and Tsn15, enzymes homologous to known [4+2] cyclases.

Purpose of the Study:

• To investigate the role of Tsn11 and Tsn15 in tetronasin biosynthesis.
• To elucidate the mechanism of tetronasin formation.
• To gain structural insights into Tsn15 activity.

Main Methods:

• Gene deletion studies (Tsn11 knockout).
• In vitro enzymatic assays and reconstitution.
• X-ray crystallography of a Tsn15-substrate complex.

Main Results:

• Deletion of Tsn11 resulted in the accumulation of an uncyclized tetronasin intermediate.
• Tsn11 catalyzes an inverse-electron-demand hetero Diels-Alder-like [4+2] cyclization.
• Tsn15 rearranges the cyclized product to form tetronasin.
• A 1.7 Å crystal structure of Tsn15 bound to its substrate was obtained.

Conclusions:

• Tsn11 and Tsn15 are essential enzymes in tetronasin biosynthesis, catalyzing distinct cyclization and rearrangement steps.
• The study reveals a novel biosynthetic pathway for tetronasin involving unexpected Diels-Alder-like chemistry.
• Structural data of Tsn15 provides mechanistic insights into its catalytic function.