Date of Award
8-7-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
In Brassica plants, glucosinolates are a diverse class of natural products that serve as plant defense molecules. Their structural diversity comes from the use of amino acids as precursors and elongation of side chains by up to nine carbons, as well as further chemical modifications. Aliphatic methionine-derived glucosinolates are the most abundant form of these compounds. Although both elongation and chemical modification of amino acid sidechains are important for methionine-glucosinolate diversity, its elongation process has not been well described. The chain elongation enzyme methylthioalkylmalate synthase (MAMS) functions as the glucosinolate pathway gatekeeper through its substrate specificity. The x-ray crystal structure of MAMS has only recently been solved and biochemical studies of the enzyme have yet to be carried out. Here we describe the studies that examine the kinetic and chemical mechanisms of Brassica juncea (Indian mustard) MAMS isoforms through initial velocity and mutagenesis studies. These studies were made possible using a 4-aldithriol-based MAMS assay that we developed. We also analyze the evolution of MAMS substrate specificity from alpha-isopropylmalate synthase (IPMS), of which MAMS is a homolog, through structural/sequence comparison methods, and discuss efforts to determine the x-ray crystallography structure of Brassica rapa IPMS. Overall, this work highlights the importance of localized structural changes in the active site for the evolution of substrate specificity in Claisen-condensing enzymes despite the conservation of chemistry. This work also provides more insights into the structure and biochemistry of MAMS, which are a useful addition to the breadth of knowledge required for efforts in metabolically engineering glucosinolates in plants and bacteria for plant defense, human health, and nutrition.
Language
English (en)
Chair and Committee
Joseph Jez
Committee Members
Hani Zaher; Robert Kranz; Tim Wencewicz; Toni Kutchan
Recommended Citation
Kitainda, Vivian, "Molecular Mechanisms Behind Methionine-Derived Glucosinolate Diversity in Brassica Plants" (2024). Arts & Sciences Electronic Theses and Dissertations. 3304.
https://openscholarship.wustl.edu/art_sci_etds/3304