Date of Award

Spring 5-10-2021

Degree Type

Thesis

Degree Name

Master of Science in Integrative Biology (MSIB)

Department

Biology

Major Professor

Susan M.E. Smith, PhD.

First Committee Member

Susan M.E. Smith, PhD.

Second Committee Member

Melanie Griffin, PhD.

Third Committee Member

Scott Nowak, PhD.

Fourth Committee Member

Austin Brown, PhD.

Abstract

NAPDH oxidase enzymes (NOXes) reduce molecular oxygen to superoxide and other ROS. NOXes contain a catalytic core comprising a heme-containing transmembrane (TM) domain and a cytoplasmic dehydrogenase (DH) domain that binds the substrate NADPH and the cofactor. Previously, NOXes were only characterized in eukaryotes, but have recently been identified in prokaryotes, namely bacteria. Due to their constitutive activity and solubility in detergent, bacterial NOXes, such as Streptococcus Pneumoniae NOX, have emerged as a model for studying NOXes. Past research studies in NOXes have identified conserved, putative interacting regions at the interface of the TM and DH domains: the TM B-loop, the TM D-loop, the NADPH-FAD linker, the TM-DH linker, and the C-terminus. Using the SpNox model system, the role of the conserved, putative interacting regions at the TM:DH interface on enzyme activity was investigated. To probe the TM:DH interface, mutations were created in the conserved regions at the TM:DH interface and peptides were designed to inhibit TM:DH interactions. Mixing experiments with separately purified TM and DH domains used heme reduction as the output to assess interruption of domain interaction by mutants or peptides. Neither the mutants nor the peptides abolished TM:DH interactions. A new full length NOX crystal structure provided an explanation for the mutants’ and peptides’ ineffectiveness. Peptide infectiveness might be explained by structural differences between SpNOX DH and eukaryotic NOX4 DH revealed by activity assays.

Available for download on Thursday, May 11, 2023

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