Modern society relies heavily on hydrocarbons. Because they are used as liquid transportation fuels, cosmetics, waxes, and food coatings, hydrocarbons are important components of most aspects of daily life. The majority of hydrocarbon products are extracted or derived from cude oil. Energy costs, fuel demand, and environmental concerns involving the non-renewable nature of petroleum-derived compounds have sparked recent interest in microbial hydrocarbon production. Engineering the diversity of microbial metabolic pathways to produce biofuels and chemicals represents a renewable alternative to fossil fuels. One pathway of interest is bacterial long-chain olefin biosynthesis. Divergent bacterial species have been shown to synthesize these waxy hydrocarbons using four enzymes: OleABCD. Recent investigations have aimed to understand how these enzymes work in concert to produce valuable hydrocarbon intermediates and products. These findings will be useful for future pathway engineering for renewable, bacterial olefin production. The first three chapters of this dissertation deal with elucidating the catalytic mechanism of the first enzyme in the olefin biosynthesis pathway, OleA. Chapters 2, 3, and 4 each investigate a separate amino acid necessary for OleA β-keto acid formation using methods of site-directed mutagenesis, biochemistry, and X-ray crystallography. In Chapter 2, the unique substrate binding channel architecture of OleA is directly demonstrated by trapping substrates and intermediates within Cys143 mutated enzymes. The role of Glu117 as the catalytic base needed to prime condensation through deprotonation of the second acyl-CoA substrate is established in Chapter 3. This represents the first dimeric thiolase superfamily enzyme that uses an active site base donated from the second monomer. It also provides evidence for the unique mechanistic strategy of OleA compared to other thiolases. Chapter 4 investigates the role His285 plays in positioning substrate and intermediates for productive condensation by OleA. It is also shown that His285 plays a role in protecting the Cys143 thiolate from oxidative damage. The dissertation concludes with the investigation of the catalytic function of OleC and the characterization of the multienzyme assembly formed by OleB, OleC, and OleD. In Chapter 6, OleC is demonstrated to produce β-lactones from β-hydroxy acids. This is the first example of a β-lactone synthetase, a novel enzyme function. It is also shown that OleC is homologous to amino acid sequences encoded in known β-lactone-producing natural product pathways, suggesting a common mechanism for β-lactone formation. Chapter 7 details the formation of an assembly consisting of OleBCD. Following co-expression of OleABCD, OleBCD are found to co-elute over nickel-affinity, anti-FLAG, and size-exclusion chromatographic purifications. These assemblies form ~2 MDa structures that produce cis-olefin following the addition of OleA and acyl-CoA. Negative stain transmission electron microscopy reveals a mixture of assemblies ranging from 24-40 nm in diameter. It is proposed that these assemblies are necessary for protecting the cell from the highly-reactive β-lactone intermediate.