Dissertation/Thesis Abstract

Function of Heme-Binding in and Native Host Expression of a Bacterial Manganese Oxidase
by Li Qian, Keizen, M.S., California State University, Fullerton, 2020, 87; 28025963
Abstract (Summary)

Microbially mediated manganese (Mn) oxidation facilitates global bioavailability of nutrients. Despite their ubiquity and appearance in diverse bacterial clades, some bacterial manganese oxidases, including MopA, remain incompletely characterized. The sequence similarity of MopA to heme peroxidases includes conserved amino acids that covalently bind heme in mammalian homologs. The nature of the bonds between MopA-hp and the heme cofactor were investigated using site-directed mutagenesis, heme staining of SDS-PAGE gels, UV-visual spectroscopy, and acetone precipitation of heterologously expressed, partially purified protein. Results indicate the conserved sites do not covalently bind heme and are not solely responsible for strong heme association. Activity assays of mutants indicate these sites contribute to manganese oxidizing activity with redundancy, though it is not yet known if the contribution is spatial or by noncovalent binding of heme. To obtain a purer MopA-hp protein preparation by avoiding contaminant Escherichia coli proteins, optimization of protein expression was attempted using the native host carrying an antibiotic-selectable broad-host-range plasmid with mopA-hp and an inducible promoter. His-tagged protein was not detected in cell lysates and in-culture activity was not significantly different between transformants and nontransformants. Colony PCR showed the plasmid insert and promoter were lost from native host transformants. Further mechanism studies and improvement of purification can contribute to understanding of this unique group of manganese oxidases.

Indexing (document details)
Advisor: Johnson, Hope A.
Commitee: Chen, Esther, Rasche, Madeline E.
School: California State University, Fullerton
Department: Biology
School Location: United States -- California
Source: MAI 82/2(E), Masters Abstracts International
Subjects: Microbiology, Geobiology, Molecular biology
Keywords: Broad-host-range plasmid, Heme, Heme peroxidase, Manganese, Manganese oxidation, Peroxidase-cyclooxygenase
Publication Number: 28025963
ISBN: 9798662585782
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