PhD Transfer Seminar – Elina Kadriu (Christendat Lab)

Investigating the function and regulatory mechanisms of microbial shikimate dehydrogenase homologs

Abstract

The shikimate pathway is required for the biosynthesis of aromatic amino acids and other aromatic compounds in bacteria. The fourth step in this pathway requires the NADPH-dependent reduction of 3-dehydroshikimate (DHS) to shikimate by the prototypical shikimate dehydrogenase (SDH) enzyme family, AroE. In addition to AroE, five other SDH family members have been identified: YdiB, Ael1, RifI, SdhL, and most recently SdhD. Previous kinetic and structural analyses of the SDH families suggests distinct biological roles not yet identified. Additionally, few studies investigate the regulation of SDH enzymes. Therefore, my research aims to investigate the biological role of the SDH family and the regulatory mechanisms which control their expression. Pseudomonas putida will be used as a model organism to investigate the functions of the SDH family of enzymes, as it has one representative from each. To characterize the RifI SDH family we investigate the role of an IclR-type transcriptional regulator (PP_2609), which we identified upstream of an uncharacterized genomic operon containing the rifI gene. To date, we have identified that PP_2609 binds to the promoter region within this operon. Additionally, the aromatic compound p-hydroxybenzoic acid (PHB) binds to the effector binding domain of PP_2609 and induces gene expression. A genomic deletion of PP_2609 exhibits improved optical density in vitro and increases lateral root counts in inoculated A. thaliana seedlings, likely due to a stress-induced morphogenic response. Moving forward, further investigations will be conducted on the molecular function of PP_2609 and the role of this operon in mitigating the phytotoxic accumulation of PHB in the rhizosphere. These analyses will uncover a novel biological role of the RifI SDH family and its mode of regulation.

The second aspect of my research is to investigate the biological role of a novel class of SDH family proteins, which we have named SdhD, from investigations of the quinate and shikimate utilization pathway in Listeria monocytogenes. Unlike other members of the SDH family, SdhD unusually redirects the NAD+-dependent oxidation of shikimate to DHS. Recently, it was shown that gallic acid (GA) produced by bacteria in the gut microbiome plays a role in facilitating colon cancer incidences in certain patients. Although the direct mechanism for gallic acid formation has not been completely elucidated, it is proposed to be produced by DHS. To investigate the role of SdhD in GA biosynthesis, I will first uncover the distribution of this protein family in the gut microbiome. Kinetic and structural analyses of SdhD will assess the conditions which facilitate GA biosynthesis. Together, this work will uncover the role of SDHs in GA biosynthesis for therapeutic potential.

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Join Zoom Meeting

https://utoronto.zoom.us/j/85901791594

Meeting ID: 859 0179 1594

Host: Dinesh Christendat (dinesh.christendat@utoronto.ca)

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