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 His10-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

pShuffle: A Plasmid for in vitro Evolution

Multi-gene shuffling is a powerful method used to combine and optimize attributes of various proteins. Here we report on the design and construction of the plasmid “pShuffle” which is suited for a variety of in vitro DNA-recombination techniques. The multiple cloning site (MCS) of pShuffle was designed to allow for the cloning of genes as well as their expression under control of either a lac- or a T7-promoter. As a specific feature, this MCS allows for the fusion of special linker sequences to both ends of cloned genes. After subsequent DNA-recombination steps, these linkers facilitate reamplification of generated gene variants, and thus may be used to construct clone libraries for activity screenings. The suitability of pShuffle for multi-gene shuffling applications was further shown with a set of styrene monooxygenase genes originating from proteo- and actinobacteria

Crystallization and preliminary characterization of chloromuconolactone dehalogenase from Rhodococcus opacus 1CP

Chloroaromatic compounds are often very persistent environmental pollutants. Nevertheless, numerous bacteria are able to metabolize these compounds and to utilize them as sole energy and carbon sources. ıt Rhodococcus opacus} 1CP is able to degrade several chloroaromatic compounds, some of them {ıt via} a variation of the 3-chlorocatechol branch of the modified {ıt ortho}-cleavage pathway. This branch in {ıt R. opacus} differs from that in {ıt Proteobacteria} in the inability of the chloromuconate cycloisomerase to dehalogenate. Instead, a unique enzyme designated as chloromuconolactone dehalogenase (ClcF) is recruited. ClcF dehalogenates 5-chloromuconolactone to {ıt cis}-dienelactone and shows a high similarity to muconolactone isomerases (EC 5.3.3.4). However, unlike the latter enzymes, it is unable to catalyse the isomerization of muconolactone to 3-⁻oxoadipate enollactone. In order to characterize the catalytic mechanism of this unusual dehalogenase, the enzyme was crystallized and subjected to X-ray structural analysis. Data sets to up to 1.65Å resolution were collected from two different crystal forms using synchrotron radiation. Crystal form I (space group {ıt P}2{\sb 1}) contained 40 subunits in the asymmetric unit, whereas ten subunits were present in crystal form II (space group {ıt P}2{\sb 1}2{\sb 1}2{\sb 1). The self-rotation function revealed the orientations of the molecular symmetry axes of the homodecamer of 52 symmetry