S94: Characterization of a fungal thioesterase having claisen cyclase and deacetylase activities in melanin biosynthesis

Melanins are a broad class of darkly-pigmented macromolecules formed by oxidative polymerization of phenolic monomers. In fungi, melanins are generally associated with the cell wall and provide survival advantages in harsh environments. They are also virulence factors for many pathogenic fungi, where pigment disruption often renders these species non-infective. The major fungal pathway for melanin biosynthesis involves polymerization of 1,8-dihydroxynaphthalene derived from 1,3,6,8-tetrahydroxynaphthalene (THN). Their backbone is assembled by members of a multidomain nonreducing polyketide synthase family, which program the synthesis of substituted aromatic products through regiospecific cyclization of linear enzyme-bound poly-β-keto intermediates fabricated from acyl-CoAs. Multiple, convergent routes to THN have evolved in fungi. Parallel heptaketide and hexaketide pathways exist that utilize separate deacylase enzymes to remove the acetoacetyl and acetyl side chains of their respective bicyclic substrates released by conventional C-terminal thioesterase/Claisen cyclase domains. In vitro characterization of Pks1 from Colletotrichum lagenarium establishes its function as a true THN synthase, unexpectedly confirming a third fungal route for synthesis of this key melanin intermediate unaided by a second enzyme. We demonstrate here that the Pks1 thioesterase (TE) is both a Claisen cyclase and a deacetylase that processes an enzyme-bound monocyclic hexaketide precursor to clarify the absence of an observable “starter-unit effect” in this system. Chimeric TE domains were generated by swapping lid regions of active sites between classes of melanin-related TEs to gain insight into this unprecedented catalysis of carbon–carbon bond making and breaking by a single α/β-hydrolase fold enzyme.