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Ho, Sui Man

University of British Columbia

Natural products containing nitrogen-nitrogen (N-N) bonds are often bioactive, such as those containing diazeniumdiolate groups. Due to the rarity of diazeniumdiolate groups in nature, their mechanisms of formation are poorly understood. Elucidation of the mechanisms employed for the biosynthesis of compounds containing N-N bonds can have applications in the fields of bioengineering and drug development. Fragin, an antimicrobial, and valdiazen, a quorum sensing signal, are two diazeniumdiolate compounds produced by the same gene cluster in Burkholderia cenocepacia H111. This thesis describes the biochemical and structural approaches I employed in the investigation of diazeniumdiolate formation in fragin and valdiazen. Nitrogen oxide species (NOx) have previously been implicated in the formation of diazeniumdiolates. Therefore, I performed chemical analyses of deletion mutants to probe for NOx production as well as to verify fragin and valdiazen production in B. cenocepacia H111. These in vivo studies revealed that a cupin protein, HamB, is non-essential and that NOx may be involved in fragin biosynthesis. Subsequent in vitro biochemical studies revealed that a diiron protein, HamA, releases NOx upon consumption of L-glutamic acid (GLU) with the assistance of HamB. Next, I performed stable isotope feeding studies with 15N-labelled substrates to elucidate the sources of nitrogen atoms in the diazeniumdiolate groups of fragin and valdiazen. These results revealed that the diazeniumdiolate group can be derived from L-valine and nitrite, which is consistent with previously proposed mechanisms. Finally, I performed structural studies of HamA to gain mechanistic insights. The X-ray crystal structure of HamA revealed a heme-oxygenase-like fold with characteristic secondary structure instability. Furthermore, analysis of the crystal structure of HamA in complex with GLU revealed that the α-amino group of GLU is proximal to the metal centre. This structural data could suggest a HamA mechanism of NOx release involving the α-amino group of GLU. Overall, this thesis has furthered our knowledge of N-N bond formation in nature. I demonstrate a route by which the diazeniumdiolate group may be formed in the fragin biosynthesis pathway, which utilizes nitrite as a precursor. I also report here the first NOx-releasing diiron enzyme, HamA, along with its crystal structure.

Text

2024-11-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/80213

Genome Science and Technology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/