Benzylsuccinate synthase

Saving the environment one molecule at a time!

 

Aromatic hydrocarbons, such as benzene, toluene and xylenes are important environmental pollutants with neurotoxic and cancer-promoting toxicity. Unfortunately, these compounds readily pollute ground water due to their high solubility, and are hard to degrade due to their aromatic nature. Remarkably, some bacteria such as Thauera aromatica are able to live on toluene as a sole carbon source, in the absence of air and using nitrate as an oxidant. The first step in anaerobic toluene metabolism is addition of toluene across the double bond of fumarate to form benzylsuccinate (shown below), which is catalyzed by benzylsuccinate synthase (BSS). Subsequent reactions result in the oxidation of benzylsuccinate to benzoylsuccinate and eventually formation of benzoyl-CoA, which is then broken down further.

We are studying BSS, the first enzyme in the toluene utilization pathway, both because of the importance of this pathway in bioremediation and because of the unusual free radical chemistry involved. Interestingly, the enzyme 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.

The mechanism of BSS

The reaction is thought to occur as shown below: the glycyl radical first removes a hydrogen a conserved cysteine residue to transiently generate a cysteinyl radical. This, in turn removes a hydrogen from the methyl group of toluene to generate a benzyl radical. The benzyl radical then undergoes a Michael-like addition to the fumarate double bond. Finally, the hydrogen is transferred back from the enzyme to generate benzylsuccinate.

We have undertaken a number of experiments using both isotopically-labeled substrates and substituted toluenes (for example p-cresol) which are also substrates for the enzyme. Using deuterated toluene we were able to show that the hydrogen atom and benzyl radical add to the same face of the double bond of fumarate. But, if maleate, the cis-isomer of fumarate, is used instead hydrogen atom and benzyl radical add to the opposite faces of the double bond. This interesting result provides evidence that benzylsuccinyl radical is formed as an intermediate: once this radical is formed, rotation about the new single bond can occur to relieve the sterically unfavorable orientation of the two carboxylate groups before the hydrogen is transferred to the product, as shown below.

 

In other experiments we have measured isotope effects to investigate the free energy profile of BSS. We have also shown that, surprisingly, the reaction can occur in reverse, to generate toluene from benzylsuccinate! The reverse reaction is only ~200-fold slower than the forward reaction, which is remarkable given the equilibrium constant favors the forward reaction by ~10¹⁰-fold.

 

 

Activation of BSS by BSS activase

We also want to investigate how the glycyl radical is formed in BSS. In related enzymes this is accomplished by an activating enzyme that uses S-adenosylmethionine and an iron-sulfur cluster to generate adenosyl radical. This radical then removes a hydrogen from the glycine residue in BSS to form the glycyl radical. We are currently working to isolate the BSS activating enzyme and study the process by which it activates BSS.

Want to know more…?

 

Recent publications from our lab on BSS

 

L. Li and E.N.G. Marsh (2006)

Mechanism of Benzylsuccinate Synthase Probed by Substrate Exchange

J. Am. Chem. Soc., 128, 16056-16058 [PDF]

L. Li and E.N.G. Marsh (2006)

Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase

Biochemistry, 45, 13932-13938 [PDF]

C. Qiao and E.N.G. Marsh (2005)

Mechanism of benzylsuccinate synthase: stereochemistry of toluene addition to fumarate and maleate

J. Am. Chem. Soc., 127, 8608-8609 [PDF]