Dimerization-Dependent Trans-Domain Coupling Enables Intermediate Transfer in Fungal Haloacid Dehalogenase-Like...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Drimane-type sesquiterpenes (DTSs) possess diverse bioactivities but their synthesis mechanisms across kingdoms are unclear.
• Haloacid dehalogenase (HAD)-like terpene cyclases (TCs) fused with phosphatases couple cyclization and dephosphorylation, yet their precise roles remain elusive.

Purpose of the Study:

• To elucidate the enzymatic mechanisms of fungal DTS formation and dephosphorylation.
• To determine the structural basis for scaffold control and phosphorylation state regulation in HAD-like TCs.

Main Methods:

• Enzyme characterization of fungal AacA as a bifunctional synthase.
• X-ray crystallography of AstC, a fungal HAD-like TC, in substrate- and product-mimic states.
• Structure-guided mutagenesis and kinetic assays with substrate analogues.

Main Results:

• AacA catalyzes farnesyl pyrophosphate cyclization and dephosphorylation.
• AstC structures reveal a homodimeric organization with inter-domain pyrophosphate transfer.
• Mutagenesis and assays identified determinants of product selectivity and dephosphorylation.

Conclusions:

• The study expands understanding of fungal DTS enzymology and HAD-like TC mechanisms.
• Structural and mechanistic insights guide future engineering of terpene cyclases for novel product synthesis.