Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

Abstract

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

Substrate range of benzylsuccinate synthase from Azoarcus sp. strain T

“…Notably, no product was observed when 4-methyl-1-cyclohexene was included in the assay rather than 1-methyl-1-cyclohexene (Table 2). This observation is consistent with the proposed radical mechanism of benzylsuccinate synthase [5,6,11], because the allyl radical intermediate that would be formed by abstraction of an H atom from the methyl carbon of 1-methyl-1-cyclohexene would be much more stable than the corresponding radical produced from 4-methyl-1-cyclohexene. Accordingly, no product was observed for methylcyclohexane (Table 2), which is fully saturated.…”

Methanogenic toluene metabolism: community structure and intermediates

“…A novel mechanism of anaerobic hydrocarbon activation, known as fumarate addition, was first discovered in studies examining the anaerobic decay of toluene by nitrate-and sulfate-reducing isolates (Biegert et al, 1996;Beller and Spormann, 1997). The genes encoding the enzyme responsible for this transformation, benzylsuccinate synthase, have been identified and sequenced in a number of bacteria (Coschigano et al, 1998;Leuthner et al, 1998;Achong et al, 2001;Winderl et al, 2007) and in a methanogenic enrichment (Washer and Edwards, 2007). Earlier investigations examining toluene biodegradation under methanogenic conditions have also implicated metabolites such as o-cresol, o-methylcyclohexanol, p-cresol, p-methylcyclohexanol, methylcyclohexane, 2-hydroxybenzoate, benzylalcohol, benzaldehyde and benzoate (Grbic'-Galic' and Vogel, 1987), suggesting that alternative mechanisms of toluene activation in the absence of endogenous electron acceptors, such as ring or methyl group hydroxylation, may be possible.…”

Construction and characterization of insertion/deletion mutations of the tutF, tutD, and tutG genes of Thauera aromatica strain T1

“…Triparental matings were carried out essentially as described previously [13,26]. Plasmids were tested for their ability to complement mutations as described previously [13].…”

“…Triparental matings were carried out essentially as described previously [13,26]. Plasmids were tested for their ability to complement mutations as described previously [13]. Toluene levels were measured by high pressure liquid chromatography using a Beckman system gold HPLC as described previously with known concentrations of toluene serving as external standards for peak identi¢cation and quanti¢cation [13].…”

“…Genes coding for this protein complex have been cloned from T. aromatica T1 (the tutFDG genes), Azoarcus sp. strain T, and T. aromatica K172 (both the bssCAB genes) [10,13,14] and transcriptional start sites for these gene complexes have been iden-ti¢ed [10,14,15]. Site-directed mutagenesis of the tutD gene from T. aromatica T1 has identi¢ed the glycine at position 828 and the cysteine at position 492 as essential for the metabolism of toluene in this strain [13].…”