Multiple C-C Bond Cleavage Reactions Catalyzed by Tolyporphin Tetrapyrrole Biosynthetic Enzymes.

Tolyporphin A is an unusual tetrapyrrole secondary metabolite containing pendant deoxysugars and unsubstituted pyrrole ß sites. Herein, we describe the biosynthesis of the tolyporphin aglycon core. HemF1 catalyzes the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III, an intermediate in heme biosynthesis. HemF2 then processes the two remaining propionate groups to generate a tetravinyl intermediate. All four vinyl groups from the macrocycle are truncated by TolI via repeated C-C bond cleavages to generate the unsubstituted pyrrole ß sites of tolyporphins. This study illustrates how the unprecedented C-C bond cleavage reactions branch from canonical heme biosynthesis to produce tolyporphins.

Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis.

Talaromyolides (1-6) are a group of unusual 6/6/6/6/6/6 hexacyclic meroterpenoids with (3R)-6-hydroxymellein and 4,5-seco-drimane substructures, isolated from the marine fungus Talaromyces purpureogenus. We have identified the biosynthetic gene cluster tlxA-J by heterologous expression in Aspergillus, in vitro enzyme assays, and CRISPR-Cas9-based gene inactivation. Remarkably, the heterodimer of non-heme iron (NHI) enzymes, TlxJ-TlxI, catalyzes three steps of oxidation including a key reaction, hydroxylation at C-5 and C-9 of 12, the intermediate with 3-ketohydroxydrimane scaffold, to facilitate a retro-aldol reaction, leading to the construction of the 4,5-secodrimane skeleton and characteristic ketal scaffold of 1-6. The products of TlxJ-TlxI, 1 and 4, were further hydroxylated at C-4'ß by another NHI heterodimer, TlxA-TlxC, and acetylated by TlxB to yield the final products, 3 and 6. The X-ray structural analysis coupled with site-directed mutagenesis provided insights into the heterodimer TlxJ-TlxI formation and its catalysis. This is the first report to show that two NHI proteins form a heterodimer for catalysis and utilizes a novel methodology to create functional oxygenase structures in secondary metabolite biosynthesis.