Structural Insight into the Reaction Mechanism of Ketosynthase-Like Decarboxylase in a Loading Module of Modular...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Ketosynthase-like decarboxylase (KS_Q) domains are vital components of modular polyketide synthases (PKSs).
• These domains are proposed to initiate polyketide synthesis by decarboxylating starter units.
• KS_Q domains share sequence similarity with ketosynthase (KS) domains but possess a Gln residue instead of Cys in their active site.

Purpose of the Study:

• To biochemically characterize KS_Q domains from FD-891 (GfsA KS_Q) and cremimycin (CmiP4 KS_Q) biosynthesis.
• To elucidate the mechanism of decarboxylation catalyzed by KS_Q domains.
• To determine the crystal structure of GfsA KS_Q and identify key catalytic residues.

Main Methods:

• In vitro biochemical assays to assess decarboxylation activity.
• X-ray crystallography to determine the structure of GfsA KS_Q with a substrate analogue.
• Site-directed mutagenesis to confirm the role of identified residues.

Main Results:

• GfsA KS_Q and CmiP4 KS_Q were confirmed to catalyze the decarboxylation of malonyl and methylmalonyl units.
• The crystal structure revealed key amino acid residues involved in the decarboxylation reaction.
• Mutational analysis validated the importance of these residues in catalysis.

Conclusions:

• A mechanism for KS_Q-catalyzed decarboxylation is proposed, involving substrate fixation and enolate formation.
• The Gln residue in KS_Q active sites mimics the acylated Cys in KS domains.
• This work provides structural and mechanistic insights into polyketide starter unit biosynthesis.