Identification of a novel self-sufficient styrene monooxygenase from Rhodococcus opacus 1CP.

Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His(10)-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

A stereoselective carbon-nitrogen lyase from Ralstonia sp. SLRS7 cleaves two of three isomers of iminodisuccinate.

Following biodegradation tests according to the OECD guidelines for testing of chemicals 301F different degradation rates were observed for the three stereoisomers of iminodisuccinate (IDS). A strain was isolated from activated sludge, which used two of three isomers, R,S-IDS and S,S-IDS, as sole source of carbon, nitrogen, and energy. The isolated strain was identified by 16S-rDNA and referred to as Ralstonia sp. SLRS7. An IDS-degrading lyase was isolated from the cell-free extract. The enzyme was purified by three chromatographic steps, which included anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The lyase catalysed the non-hydrolytic cleavage of IDS without requirement of any cofactors. Cleavage of S,S-IDS led to the formation of fumaric acid and L-aspartic acid. Interestingly R,S-IDS yielded only D-aspartic acid besides fumaric acid. R,R-IDS was not transformed. Thus, the IDS-degrading enzyme is a carbon-nitrogen lyase attacking only the asymmetric carbon atom exhibiting the S-configuration. Besides S,S-IDS and R,S-IDS cleavage, the lyase catalysed also the transformation of certain S,S-IDS metal complexes, namely Ca(2+)-, Mg(2+)- and Mn(2+)-IDS. The maximum enzyme activity was found at pH 8.0-8.5 and 35 degrees C. SDS-PAGE analysis revealed a single 57-kDa protein band. The native enzyme was estimated to be around 240 kDa indicating a homotetramer enzyme.

A linear megaplasmid, p1CP, carrying the genes for chlorocatechol catabolism of Rhodococcus opacus 1CP.

The Gram-positive actinobacterium Rhodococcus opacus 1CP is able to utilize several (chloro)aromatic compounds as sole carbon sources, and gene clusters for various catabolic enzymes and pathways have previously been identified. Pulsed-field gel electrophoresis indicates the occurrence of a 740 kb megaplasmid, designated p1CP. Linear topology and the presence of covalently bound proteins were shown by the unchanged electrophoretic mobility after S1 nuclease treatment and by the immobility of the native plasmid during non-denaturing agarose gel electrophoresis, respectively. Sequence comparisons of both termini revealed a perfect 13 bp terminal inverted repeat (TIR) as part of an imperfect 583/587 bp TIR, as well as two copies of the highly conserved centre (GCTXCGC) of a palindromic motif. An initial restriction analysis of p1CP was performed. By means of PCR and hybridization techniques, p1CP was screened for several genes encoding enzymes of (chloro)aromatic degradation. A single maleylacetate reductase gene macA, the clc gene cluster for 4-chloro-/3,5-dichlorocatechol degradation, and the clc2 gene cluster for 3-chlorocatechol degradation were found on p1CP whereas the cat and pca gene clusters for the catechol and the protocatechuate pathways, respectively, were not. Prolonged cultivation of the wild-type strain 1CP under non-selective conditions led to the isolation of the clc- and clc2-deficient mutants 1CP.01 and 1CP.02 harbouring the shortened plasmid variants p1CP.01 (500 kb) and p1CP.02 (400 kb).