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Hedges, Jason

University of British Columbia

Over time, evolution can result in enzymes developing novel functions. An emerging example of this phenomenon comes from studies of pyridoxal-5ʹ-phosphate (PLP)-dependent enzymes, which catalyze a diverse set of chemical reactions on amino acid substrates. This thesis describes the discovery and characterization of some unusual PLP-dependent enzymes. Some PLP-dependent enzymes have been shown to catalyze challenging oxidations of an L-arginine substrate using O₂ as a co-substrate. In Chapter 2 I set out to describe new PLP-dependent arginine oxidases and study how they function. Using bioinformatics, I was able to hone in on one particular enzyme, named RohP. Biochemical characterization of RohP revealed that it is an arginine hydroxylase, which catalyzes the formation of (S)-4-hydroxy-2-ketoarginine. Furthermore, I was able to obtain several high-resolution X-ray crystal structures of RohP at different stages of its catalytic cycle. Together these results advance the understanding of how O₂- and PLP-dependent enzymes function. RohP was found in a conserved five gene biosynthetic gene cluster, with no known product. Therefore, in Chapter 3 I set out to determine what the product of this unusual biosynthetic gene cluster was. Using the studies of RohP as a starting point, additional in vitro biochemical investigations of four other enzymes encoded along with RohP in this biosynthetic gene cluster revealed that together they convert L-arginine to the antibiotic azomycin (2-nitroimidazole). As azomycin was first isolated over 50 years ago, the discoveries described in this chapter solve a longstanding biosynthetic mystery. Interesting PLP-dependent enzymes are found in many biosynthetic pathways. In Chapter 4 I report my characterization of BesB, an unrelated PLP-dependent enzyme which catalyzes the formation of a terminal alkyne bond. BesB has limited solubility in E. coli, which has hampered its study initially. Through use of a different heterologous expression system I was able to obtain soluble BesB. Through biochemical and X-ray crystallographic analysis, an active site phenylalanine substitution appears to be key to unlocking the novel reactivity of BesB. This study provides the first crystal structures of any alkyne-forming enzyme. Insights from the studies of RohP and BesB should prove useful in developing novel PLP-dependent biocatalysts.

Text

2024-07-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/79048

Chemistry

University of British Columbia

UBCV

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