STRUCTURE-FUNCTION ANALYSIS AND ENGINEERING OF BIOSYNTHETIC ENZYMES

Takahiro Mori | Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan,

Natural products exhibit remarkable structural diversity and include numerous bioactive compounds utilized in pharmaceuticals, food, and food additives. Our research has focused on elucidating the mechanisms by which the structural diversity and complexity of secondary metabolites are constructed in nature through the discovery of biosynthetic genes, the biophysical and biochemical characterization of enzymes, and the structural analysis of enzyme–enzyme interactions. Furthermore, we have explored the generation of molecular diversity through enzyme engineering and synthetic biology.

Lincosamides, isolated from Streptomyces species, are thiooctose-containing natural products that are a class of clinically used antibiotics.[1] The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal 5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyzes thiol formation through β-elimination while CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative-deamination. The structural basis for the diversification mechanism remains largely unexplored. Here, we conducted structure-function analyses of LmbF and CcbF. The X-ray crystal structures, docking, and molecular dynamics simulations revealed that active site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide. [2]

[1] Janata, J., et al., Nat Prod Rep 35, 257 (2018).

[2] Mori, T., et al., Nature Chemistry, 17, 256 (2025).

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Date: May 09, 2025 | 01:30 PM - 04:00 PM

Venue: University of Science, 227 Nguyen Van Cu

Room: I.35

Register here: bit.ly/25fbbseminar01