Polyketide synthases
(PKSs) are versatile C–C bond-forming
enzymes that are broadly distributed in bacteria and fungi. The polyketide
compound family includes many clinically useful drugs such as the
antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering
lovastatin. Harnessing PKSs for custom compound synthesis remains
an open challenge, largely because of the lack of knowledge about
key structural properties. Particularly, the domains—well characterized
on their own—are poorly understood in their arrangement, conformational
dynamics, and interplay in the intricate quaternary structure of modular
PKSs. Here, we characterize module 2 from the 6-deoxyerythronolide
B synthase by small-angle X-ray scattering and cross-linking mass
spectrometry with coarse-grained structural modeling. The results
of this hybrid approach shed light on the solution structure of a
cis-AT type PKS module as well as its inherent conformational dynamics.
Supported by a directed evolution approach, we also find that acyl
carrier protein (ACP)-mediated substrate shuttling appears to be steered
by a nonspecific electrostatic interaction network.
Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.
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