Crystallographic and biochemical characterization of key steps in reductasporine and capuramycin biosynthesis
department or school: Science, Faculty of
department or school: Chemistry, Department of
data provider: DSpace
degree campus: UBCV
creator: Daniel-Ivad, Phillip
issued: 2024-02-05T17:15:10Z
issued: 2024
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: 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 P…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/87410
note: CRYSTALLOGRAPHIC AND BIOCHEMICAL CHARACTERIZATION OF KEY STEPS IN REDUCTASPORINE AND CAPURAMYCIN BIOSYNTHESIS by Phillip Daniel-Ivad B.Sc., McMaster University, 2014 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February 2024 © Phillip Daniel-Ivad, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Crystallographic and biochemical characterization of key steps in reductasporine and capuramycin biosynthesis submitted by Phillip Daniel-Ivad in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry Examining Committee: Katherine Ryan, Professor, Chemistry, UBC Supervisor Martin Tanner, Professor, Chemistry, UBC Supervisory Committee Member Harry Brumer, Professor, Chemistry, UBC Supervisory Committee Member David Perrin, Professor, Chemistry, UBC University Examiner Filip van Petegem, Professor, Biochemistry & Molecular Biology, UBC University Examiner Additional Supervisory Committee Members: Marco Ciufolini, Professor Emeritus, Chemistry, UBC Supervisory Committee Member iii Abstract 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 prov…
has type: Thesis/Dissertation
date issued: 2024-05
is shown at: 10.14288/1.0439615
language: eng
degree discipline: Chemistry
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Ryan, Katherine S.
type: Text
identifier URI: http://hdl.handle.net/2429/87410