Fe(II)/2-oxoglutarate-dependent dioxygenase bifunctionally catalyzes ring formation involving sequential terminal carbon oxidation for pyrrolizidinone alkaloid biosynthesis.
Abstract
The Fe(II)/2-oxoglutarate-dependent dioxygenase UcsF plays a key role in the early biosynthesis of UCS1025A, an antibacterial and antitumor pyrrolizidinone alkaloid, by participating in the formation of its core ring scaffold. UcsF catalyzes sequential oxidation of the terminal C5 of L-isoleucine, ultimately generating a five-membered ring product, (2S,3S)-3,4-dihydro-3-methyl-2H-pyrrole-2-carboxylic acid. This catalytic process is vital in the construction of natural product skeletons and synthetic biology. However, the molecular basis of UcsF and its unique two-step catalytic mechanism remain poorly understood. We characterized the catalytic function of UcsF, determined its crystal structure and combined molecular docking and MD simulations to reveal the molecular basis for substrate recognition, and employed quantum chemical calculations on reaction-state models to investigate plausible reaction pathways underlying the sequential oxidation. UcsF exhibits catalytic activity toward 2-amino carboxylic acid substrates. The crystal structure of UcsF features a unique open active pocket, with substrate binding defined by the collective contributions of multiple structural modules. In the catalytic reaction, the Fe(IV) = O species abstracts a hydrogen atom from the C5 position of L-isoleucine, followed by hydroxyl rebound to complete the first hydroxylation step. Combined kinetic, solvent isotope-effect, mutational, and computational analyses support a preferred Asp279'-facilitated PCET pathway for the second oxidation of 5-OH-Ile to the aldehyde product. Our study provides a molecular basis for understanding the relationship between the structure and function of Fe(II)/2-oxoglutarate-dependent dioxygenases and reveals the potential of the catalytic ability of UcsF for biosynthesis.
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