Mechanistic Investigation and Engineering of a Membrane-Bound Hydrocarbon-Producing Metalloenzyme

Abstract

The pursuit of sustainable and environmentally friendly fuels, such as biofuels, is gaining attention as an alternative to diminishing fossil fuel resources. Consequently, there is a significant focus on the sustainable biosynthesis of hydrocarbons since these molecules are the primary components of fossil fuels. Hydrocarbons, such as 1-alkenes, also play a pivotal role in various industries, serving as essential components in producing polymers, lubricants, and detergents. In nature, 1-alkenes are produced from naturally abundant fatty acids by enzymes known as fatty acid decarboxylases. My research is focused on UndB, the only known membrane-bound fatty acid decarboxylase with unparalleled potential for catalyzing the direct conversion of free fatty acids into 1-alkenes. Despite its promise, the molecular intricacies of UndB have remained poorly understood until now. In this thesis, we report the purification and unraveling of the catalytic mysteries of UndB for the first time. Our study establishes UndB as a non-heme diiron-enzyme utilizing a conserved histidine cluster at the active site. We also decipher the dependency of UndB on molecular oxygen and reductants and identify the enzyme's optimal redox partners. We determine the kinetic parameters of UndB and demonstrate that UndB prefers medium-chain fatty acids as substrate. By delving into the cryptic catalysis of UndB at the membrane interface, we provide evidence supporting a hydrogen atom transfer (HAT)-mediated initiation of the reaction. Kinetic isotopic effect (KIE) experiments demonstrate a significant KIE for the abstraction of the C3 hydrogen of the substrate, and investigations into C1−C2 bond cleavage suggest a HAT-mediated mechanism. Building upon understanding the catalytic mechanisms, we address limited turnovers of UndB observed due to peroxide-mediated rapid inactivation of the enzyme during catalysis. Through strategic engineering, we create a chimeric membrane enzyme by genetically fusing UndB with catalase, mitigating peroxide-induced inactivation and significantly enhancing its total turnovers. The chimeric enzyme stands as a pioneering example of successful membrane enzyme engineering with catalase. To substantiate the practical applications of UndB for efficient 1-alkene biosynthesis, we develop a novel whole-cell biocatalyst, achieving exceptional efficiencies (up to 95%) in the biotransformation of diverse fatty acids (aliphatic as well as aromatic) into corresponding 1-alkenes. This breakthrough marks a significant stride in the potential use of 1-alkenes for biofuel and green commodity chemical production, utilizing naturally abundant resources. In conclusion, the research work presented in this thesis sheds light on the molecular intricacies of UndB catalysis and advances our understanding of hydrocarbon biosynthesis in nature. By providing key insights into the catalytic mechanism of UndB, strategically engineering the enzyme, and making it ready for the biosynthesis of a broad range of 1-alkenes, this research contributes to the development of sustainable alternatives and biotechnological solutions, propelling us toward a greener and more environmentally conscious future.