IDENTIFICATION AND ANALYSIS OF GENES INVOLVED IN ANAEROBIC TOLUENE METABOLISM BY STRAIN T1 - PUTATIVE ROLE OF A GLYCINE FREE-RADICAL

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene ut ilization 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. The tutD gene codes for an 86 4-amino-acid protein with a calculated molecular mass of 97,600 Da, Th e tutE gene codes for a 375-amino-acid protein with a calculated molec ular mass of 41,300 Da, Two additional small open reading frames have been identified, but their role is not known. The TutE protein has hom ology to pyruvate formate-lyase activating enzymes. The TutD protein h as homology to pyruvate formate-lyase enzymes, including a conserved c ysteine residue at the active site and a conserved glycine residue tha t is activated to a free radical in this enzyme. Site-directed mutagen esis of these two conserved amino acids shows that they are also essen tial for the function of TutD.