Biochemical studies on Pseudomonas spp. ethylene-forming enzymes

Thesis

2-oxoglutarate (2OG)-dependent oxygenases are non-haem Fe(II) enzymes that catalyse a diverse repertoire of oxidative transformations. The ethylene-forming enzyme from Pseudomonas savastanoi (PsEFE) catalyses a bifurcating reaction: the C5 hydroxylation of Larginine coupled to the oxidative decarboxylation of 2OG to succinate and the conversion of 2OG to ethylene and CO2. This thesis presents biochemical studies on PsEFE and two other plant oxygenases, HIS1 and OsHSL1, involved in conferring herbicide resistance in plants; the research described in this thesis explores the potential applications of 2OG oxygenases in biocatalysis and agriculture.

Chapter 1 introduces 2OG oxygenases and ethylene biosynthesis in a comprehensive literature review. Chapter 2 discuses substrate scope studies on PsEFE and explores how substitution patterns of 2OG derivatives affect reaction outcomes. Chapter 3 validates EFEs from two additional P. savastanoi strains, 1449B and glycinea PsEFE; this chapter also reveals a crystallographically-observed atypical binding mode of 2OG in 1449B PsEFE.

Chapter 4 expands the work on PsEFE and 2OG derivatives, describing PsEFE active site variants that enable the production of alkenes other than ethylene from substituted 2OG derivatives. Chapter 5 investigates the interaction of PsEFE with hydrophobic 2-oxoacids, derived from proteinogenic and non-proteinogenic amino acids. This chapter also reports on PsEFE active site variants that enable ethylene production from 2-keto-4-methylthiobutyrate (KMBA) and 2-keto-4-ethylthiobutyrate (KEBA).

Chapter 6 describes biochemical and structural studies of HIS1 and OsHSL1, herbicide resistance enzymes from Oryza sativa, which confer resistance to widely-used β-triketone herbicides via hydroxylation. This work highlights how catalytic promiscuity in the form of resistance poses challenges to global food security. Chapter 7 summarises the results and discusses the biocatalytic potential and promiscuity of 2OG oxygenases.

Collectively, the work described in this thesis advances the biochemical understanding of PsEFE and discusses how the (co)substrate structure and active-site architecture govern reaction partitioning. Guided by substrate scope analyses, targeted mutagenesis, and structural characterisation, this work demonstrates how PsEFE reactivity has the potential to be tuned, improved, and harnessed for biocatalytic and biotechnological applications.

Authors: Dhingra, S

• DOI: 10.5287/ora-doxookpge
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Bioinorganic chemistry; Chemistry