Characterisation of exceptional ring-cleaving dioxygenases
In previous studies, we genetically and biochemically characterised three different ring-cleaving extradiolic dioxygenases from S. xenophaga BN6 and were able to demonstrate ring cleavage for a monohydroxylated aminosalicylate for the first time (Kuhm et al., 1991; Stolz et al., 1992; Stolz & Knackmuss, 1993a; Heiss et al., 1995, 1997). Here, an unusual ring-cleaving dioxygenase was identified in S. xenophaga BN6, which differed from all previously known extradiolic dioxygenases with respect to the size of the subunits and the structure of the holoenzyme. Surprisingly, this enzyme oxidised 3-chloro pyrocatechol by a previously undescribed distal extradiolic ring cleavage (Heiss et al., 1995). For this enzyme, we first determined basic biochemical parameters and cloned the corresponding gene.
In this system, the ability to turnover 3-chloro pyrocatechol could be significantly increased by targeted and untargeted mutagenesis techniques. In the course of these mutagenesis experiments, a change in the ring cleavage direction of extradiolic dioxygenases was demonstrated for the first time and the classical proximal ring cleavage of alkylpyrocatechins was modified to a distal ring cleavage mechanism (Riegert et al., 1998, 1999, 2001).
In further studies, the exceptional 4-sulfocatechol converting protocatechuate-3,4-dioxygenases from the sulphanilic acid degrading mixed culture were investigated. For this purpose, the type I and type II genes of the protocatechuate-3,4-dioxygenases were cloned from the strains H. intermedia S1 and A. radiobacter S2 (Contzen & Stolz, 2000; Contzen et al., 2001). By comparing the amino acid sequences of the two P34OIIs from strains S1 and S2 with sequences of type I enzymes from other bacterial strains already published in the literature and using known structural data for the catalytic centre of P34O (type I) from Pseudomonas putida, the amino acid residue tryptophan(ß149) in the catalytic centre could be identified, which was conserved in all known type I P34Os, but was replaced in the two P34O-IIs by a sterically less demanding amino acid (valine or isoleucine). isoleucine).
The targeted replacement of this tryptophan residue in the P34O-I of strain S2 with a valine residue allowed the mutant form of the enzyme to turnover 4-sulfocatechol. This provided the first molecular biological evidence for the basis of the adaptation of enzymes to the turnover of sulphonated compounds foreign to nature (Contzen et al., 2001).
Another unusual ring-cleaving dioxygenase was discovered in the 6-aminonaphthalene-2-sulfonic acid-degrading bacterial strain Pseudaminobacter salicylatoxidans BN12. This enzyme has the previously undescribed ability to oxidise the monohydroxylated substrate salicylate (2-hydoxybenzoate) by a direct cleavage of the aromatic ring to 2-oxohepta-3,5-dienoate. The reaction product was identified beyond doubt by spectrometric and spectroscopic techniques (Hintner et al., 2001). The ring-cleaving dioxygenase was purified to homogeneity and it was shown that the purified enzyme converted not only salicylate but also various substituted salicylates (e.g. 3-, 4- or 5-chloro or methyl salicylate). The coding gene was cloned, sequenced and functionally overexpressed in E. coli. Sequence comparisons showed that the "salicylate-1,2-dioxygenase" is apparently related to gentisate-1,2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases.
By analysing the oxidation of different substituted salicylates using HPLC/MS techniques, a dehalogenation mechanism resulting from intramolecular lactonisation could be demonstrated for the turnover in the 5-position of halogenated salicylates (Hintner et al., 2004). The crystal structure of the enzyme was elucidated in a collaboration with the working group of Prof. F. Briganti (University of Florence) (Matera et al. 2008) and the molecular basis of the extraordinary substrate specificity was analysed with the help of various mutants (Steimer, 2009).
Publications of analysis and modification of ring-cleaving dioxygenases
Other research fields:
Biodegradation of azo dyes and sulphonated aromatics