cAD and BSS

Our laboratory is interested in enzymes that are involved in the biosynthesis and degradation of hydrocarbons. Hydrocarbon-degrading enzymes have potential applications in bioremediation and hydrocarbon-synthesizing enzymes provide pathways for the biosynthesis of next-generation biofuels. In addition, these enzymes catalyze chemically unusual reactions by mechanisms that are not understood. We are currently investigating two unusual enzymes benzylsuccinate synthase, which catalyzes the first step toluene degradation by some anaerobic bacteria, aldehyde decarbonylase that catalyzes the last step in alkane biosynthesis in plants, insects and cyanobacteria.

Aldehyde decarbonylase

Although the plant and insect enzymes are integral membrane proteins, the cyanobacterial aldehyde decarbonylase (cAD) is, surprisingly, a soluble protein whose structure is related to that of methane mono-oxygenase. We recently showed that unlike the membrane-bound enzymes, which produce CO or CO₂ as a side product, cAD instead converts the aldehyde carbon to formate in a reaction that requires iron, a reducing system and oxygen. We are working to understand how the enzyme catalyzes this unusual reaction that we believe involves a radical mechanism.

Benzylsuccinate synthase

Benzylsuccinate synthase (BSS) catalyzes the first step in anaerobic toluene metabolism is addition of toluene across the double bond of fumarate to form benzylsuccinate. Subsequent reactions result in the oxidation of benzylsuccinate to benzoylsuccinate and eventually formation of benzoyl-CoA, which is then broken down further. This highly unusual reaction is thought to share mechanistic similarities with anaerobic ribonucleotide reductase and pyruvate formate-lyase, although these enzymes catalyze very different chemical reactions. Each of these enzymes contains an organic radical located on specific glycine residue within the protein. One challenge is to find out how these enzymes use the same radical to initiate such different chemical reactions.

Experiments using both isotopically-labeled substrates and substituted toluenes, which are also substrates for the enzyme, suggest a mechanism involving the formation of a benzyl radical that then adds across the double bond of fumarate. Most recently we established that the enzyme also contains two iron sulfur clusters, whose functions are currently unknown. Our current efforts are focused on determining the crystal structure of the enzyme.