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Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits, alpha, beta, and gamma, that in Thauera aromatica strain T1 are encoded by the tutD, tutG, and tutF genes, respectively. The large alpha-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small beta- and gamma-subunits has remained unclear. We have overexpressed all three subunits of benzylsuccinate synthase in Escherichia coli, both individually and in combination. Coexpression of the gamma-subunit (but not the beta-subunit) is essential for efficient expression of the alpha-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an alpha(2)beta(2)gamma(2) hexamer by nickel affinity chromatography through a "His(6)" affinity tag engineered onto the C-terminus of the alpha-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the beta-subunit and the other with the gamma-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly, a mutant gamma-subunit lacking the [4Fe-4S] cluster is unable to stabilize the alpha-subunit when the proteins are coexpressed.

Benzylsuccinate synthase, encoded by the tutF, tutD, and tutG genes of Thauera aromatica strain T1, is responsible for the first step of anaerobic toluene metabolism. Previous work has shown that these genes are part of the tutE tutFDGH gene cluster and strains carrying a mutation in the tutE, tutF, tutD, or tutG genes are unable to metabolize toluene. In this study, we performed site-directed mutagenesis of the tutE, tutF, and tutG genes and determined that the cysteines at position 72 and 79 of TutE are likely to be critical for the radical activation of benzylsuccinate synthase, while the cysteine alanine at positions 9 and 10 of TutF, and the cysteine at position 29 of TutG are also essential for toluene metabolism. Additionally, we report that the tutH gene is necessary for toluene metabolism and the glycine lysine serine (part of the putative ATP/GTP binding domain) at positions 52-54 of the TutH protein is essential for toluene metabolism.

Expression of the tutE tutFDGH gene cluster of Thauera aromatica strain T1 was examined by Northern and Western analysis in a wild-type strain and chromosomally deleted strains with or without in-frame deletion plasmids. While expression was observed when the wild-type strain was induced with toluene, various chromosomally deleted strains exhibited little or no expression of the tut genes. In contrast, both wild-type and chromosomally deleted strains expressed the tut genes when induced with benzylsuccinate. We conclude that benzylsuccinate is required for the full induction of the tutE tutFDGH gene cluster of T. aromatica strain T1.

Thauera aromatica T1 was isolated for its ability to use toluene as a sole carbon source under denitrifying conditions. A genetic approach was used to examine the roles of the tutF, tutD, and tutG gene products (part of a single operon) in the metabolism of toluene. The genes were individually deleted from the chromosome and each resulting mutant strain was unable to metabolize toluene. Plasmids carrying individual in-frame gene deletions failed to complement the corresponding chromosomal deletions but did complement chromosomal deletions downstream of the in-frame deletion. Hence, the tutF, tutD, and tutG genes are each essential for toluene metabolism in T. aromatica T1.

The denitrifying strain T1, identified as Thauera aromatica, is able to grow with toluene serving as its sole carbon source. Previous work identified two genes, tutD and tutE, that are involved in toluene metabolism. Two small open reading frames, tutF and tutG, which may also play a role in toluene metabolism, were also identified. The present work examines the transcriptional organization and regulation of these toluene utilization genes. Northern analysis indicates that the four genes are organized into two operons, tutE and tutFDG, and that both operons are regulated in response to toluene. Primer extension analysis has identified major transcriptional start sites located 177 bp upstream of the tutE translational start and 76 bp upstream of the tutF translational start. Furthermore, a fifth gene, tutH, has been identified immediately downstream of tutG. It is transcribed from the same start site as tutFDG and is predicted to code for a 286-amino-acid protein with a calculated molecular mass of about 31,800 Da. The TutH protein is predicted to have an ATP/GTP binding domain and is similar to the NorQ/NirQ family of proteins.