Growth of Aquincola tertiaricarbonis L108 on tert‐Butyl Alcohol Leads to the Induction of a Phthalate Dioxygenase‐related Protein and its Associated Oxidoreductase Subunit

Abstract

AbstractThe microbial degradation of tert‐butyl alcohol (TBA), an important environmental pollutant and an intermediate in the degradation of methyl tert‐butyl ether (MTBE), was proposed to involve a monooxygenase for the initial oxidation of TBA, but up to now a specific enzyme with that activity has not been described except the well‐known AlkB for the Gram‐positive strain Mycobacterium austroafricanum IFP2012 (Lopez Ferreira et al., Appl. Microbiol. Biotechnol. 2007, 75, 909–919). In the course of our studies of the MTBE pathway, the proteome patterns in one‐ and two‐dimensional gels of Aquincola tertiaricarbonis L108 which was grown on lactate, on hydroxyisobutyrate (2‐HIBA) and TBA, were compared. A protein of about 55 kDa was detected after growth on TBA and 2‐HIBA, which, after mass spectrometric analysis of the tryptic digested peptides, was assigned with a high score to phthalate dioxygenase. Sequence analysis of PCR products obtained with primers derived from the amino acid sequences in the above peptides supported the assignment to the hydroxylase subunit of phthalate dioxygenase‐like proteins by covering 96.7 % of a corresponding gene from Methylibium petroleiphilum PM1. The conserved amino acid motifs ‐R‐x12‐CxHRxxxLxxG‐x8‐CxYHR‐x6‐G‐ for the Rieske [2Fe‐2S] binding domain and (‐D/E)xxxDxxHxxxxH‐ for the mononuclear iron binding domain were found. A second protein of about 38 kDa was detected after growth on TBA with a lower score and attributed to a putative iron‐sulfur oxidoreductase subunit. Primers derived from the peptides resulted in an amplicon, which covered 75.7 % of a corresponding gene from M. petroleiphilum PM1. Conserved motifs ‐RxYSL‐x20‐22‐RGGS‐ for FMN binding and ‐GGIGxTPxxxM‐ for NAD binding were detected, which suggests that this protein is the small subunit of a two‐component phthalate dioxygenase‐like enzyme typically containing FMN. Dioxygenase‐related enzymes are known to catalyze also monooxygenase reactions (see e.g. Zhou et al. J. Bacteriol. 2002, 184, 1547–1555), which makes it likely that the two proteins induced in the presence of TBA are involved in TBA oxidation.