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Daniel-Ivad, Phillip

University of British Columbia

Studying individual biosynthetic transformations in the creation of natural products often reveals surprising and powerful chemical reactivities and molecular handling strategies. Indolocarbazole bisindoles have been widely tested in clinical studies and arise from oxidative dimerization of L-tryptophan. Among bisindoles, reductasporine bears an unusual dimethylpyrrolinium structure. Its biosynthesis differs from other indolocarbazole pathways by two tailoring enzymes: the imine reductase RedE and N,N-dimethyltransferase RedM. Here I reconstitute this pathway in vitro and show that RedE prevents unstable indolocarbazole intermediates from becoming oxidized and provides reduced didemethylreductasporine substrate to RedM. Employing X-ray crystallography, I solved two ternary complexes of RedE co-crystallized with the substrate-mimic arcyriaflavin A, revealing an extended active site cleft with distinct secondary indolocarbazole binding site. Site-directed mutagenesis confirms the conserved active site aspartate (D168) is essential for activity and anchors the substrate via hydrogen-bonding. Variants targeting the secondary binding site reduce catalytic efficiency, suggesting this site protects the substrate from autooxidation. I solved the 1.7 Å structure of RedM demonstrating it adopts distinct open and closed conformations with either SAH or SAM cofactor, respectively. Site-directed mutagenesis, docking and sequence bioinformatics identify conserved substrate-recognizing residues and suggest dimethyltransferase catalytic activity likely arises from precise orientation and desolvation of the substrate. Recently, Cap15 has been shown to be an oxygen- and pyridoxal phosphate (PLP)-dependent enzyme and is the first example of this activity in the L-seryl-tRNA(Sec) selenium transferase enzyme family. It catalyzes oxidative-decarboxylation of 5ʹ-glycyl uridine to the corresponding 5ʹ-carboxamide uridine. Solving for the 2.40 Å resolution crystal structure of Cap15 shows PLP bonds to K230 and a phosphate anion in the active site bridges N- and C-terminal domains. Sequence analysis reveals the loop proximal to the internal aldimine and hydrogen bonding to the active site phosphate is strictly conserved among Cap15 homologues present in capuramycin-type gene clusters. The crystal structures provide a basis for further investigations into the sequence-determinants of secondary binding site formation in RedE, dimethylation activity in RedM and oxygen-consuming activity in Cap15. RedE in particular can serve as a starting-point in the engineering of imine reductases to accommodate large substrates in the production of industry-relevant chiral amines.

Text

2024-02-05

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/87410

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/