Recombinant expression of a unique chloromuconolactone dehalogenase ClcF from Rhodococcus opacus 1CP and identification of catalytically relevant residues by mutational analysis

2012 ◽  
Vol 526 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Janosch A.D. Gröning ◽  
Christian Roth ◽  
Stefan R. Kaschabek ◽  
Norbert Sträter ◽  
Michael Schlömann
Keyword(s):  
Mutational Analysis ◽  
Recombinant Expression ◽  
Rhodococcus Opacus ◽  
Rhodococcus Opacus 1Cp

Characterization of the Maleylacetate Reductase MacA of Rhodococcus opacus 1CP and Evidence for the Presence of an Isofunctional Enzyme

1998 ◽  
Vol 180 (14) ◽  
pp. 3503-3508 ◽  
Author(s):  
Volker Seibert ◽  
Elena M. Kourbatova ◽  
Ludmila A. Golovleva ◽  
Michael Schlömann
Keyword(s):  
Aromatic Compounds ◽  
Gene Clusters ◽  
Reductive Dehalogenation ◽  
Rhodococcus Opacus ◽  
Reductase Gene ◽  
Rhodococcus Opacus 1Cp ◽  
Dienelactone Hydrolase ◽  
Maleylacetate Reductase ◽  
Intradiol Dioxygenase

ABSTRACT Maleylacetate reductases (EC 1.3.1.32 ) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene macA, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain Rhodococcus opacus 1CP. Sequencing of macA showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned Rhodococcusmaleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of macA, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with macA encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the R. opacus maleylacetate reductase genemacA clearly is not part of a specialized chlorocatechol gene cluster.

Identification and structural characterisation of novel trehalose dinocardiomycolates from n-alkane-grown Rhodococcus opacus 1CP

2005 ◽  
Vol 70 (5) ◽  
pp. 605-611 ◽  
Author(s):  
Susanne Niescher ◽  
Victor Wray ◽  
Siegmund Lang ◽  
Stefan R. Kaschabek ◽  
Michael Schlömann
Keyword(s):  
Rhodococcus Opacus ◽  
Structural Characterisation ◽  
Rhodococcus Opacus 1Cp

scholarly journals Catalytic and hydrodynamic properties of styrene monooxygenases from Rhodococcus opacus 1CP are modulated by cofactor binding

AMB Express ◽  
2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Anika Riedel ◽  
Thomas Heine ◽  
Adrie H Westphal ◽  
Catleen Conrad ◽  
Philipp Rathsack ◽  
...  
Keyword(s):  
Rhodococcus Opacus ◽  
Hydrodynamic Properties ◽  
Cofactor Binding ◽  
Rhodococcus Opacus 1Cp

Metabolism of 3-hydroxybenzoate and gentisate by strain Rhodococcus opacus 1CP

Microbiology ◽  
2012 ◽  
Vol 81 (3) ◽  
pp. 299-305 ◽  
Author(s):  
N. M. Subbotina ◽  
M. P. Kolomytseva ◽  
L. A. Golovleva
Keyword(s):  
Rhodococcus Opacus ◽  
Rhodococcus Opacus 1Cp

scholarly journals Evaluation of 3-Chlorobenzoate 1,2-Dioxygenase Inhibition by 2- and 4-Chlorobenzoate with a Cell-Based Technique

Biosensors ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 106 ◽  
Author(s):  
Emelyanova ◽  
Solyanikova
Keyword(s):  
Competitive Inhibition ◽  
Enzymatic Reaction ◽  
Oxygen Electrode ◽  
Electrochemical Reactor ◽  
Rhodococcus Opacus ◽  
Microbial Sensor ◽  
Rhodococcus Opacus 1Cp ◽  
Excess Substrate ◽  
A Cell ◽  
The Hill

The electrochemical reactor microbial sensor with the Clark oxygen electrode as the transducer was used for investigation of the competition between 3-chlorobenzoate (3-CBA) and its analogues, 2- and 4-chlorobenzoate (2-CBA and 4-CBA), for 3-chlorobenzoate-1,2-dioxygenase (3-CBDO) of Rhodococcus opacus 1CP cells. The change in respiration of freshly harvested R. opacus 1CP cells in response to 3-CBA served as an indicator of 3-CBDO activity. The results obtained confirmed inducibility of 3-CBDO. Sigmoidal dependency of the rate of the enzymatic reaction on the concentration of 3-CBA was obtained and positive kinetic cooperativity by a substrate was shown for 3-CBDO. The Hill concentration constant, S0.5, and the constant of catalytic activity, Vmax, were determined. Inhibition of the rate of enzymatic reaction by excess substrate, 3-CBA, was observed. Associative (competitive inhibition according to classic classification) and transient types of the 3-CBA-1,2-DO inhibition by 2-CBA and 4-CBA, respectively, were found. The kinetic parameters such as S0.5i and Vmaxi were also estimated for 2-CBA and 4-CBA. The disappearance of the S-shape of the curve of the V versus S dependence for 3-CBDO in the presence of 4-CBA was assumed to imply that 4-chlorobenzoate had no capability to be catalytically transformed by 3-chlorobenzoate-1,2-dioxygenase of Rhodococcus opacus 1CP cells.

scholarly journals Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

2009 ◽  
Vol 191 (15) ◽  
pp. 4996-5009 ◽  
Author(s):  
Dirk Tischler ◽  
Dirk Eulberg ◽  
Silvia Lakner ◽  
Stefan R. Kaschabek ◽  
Willem J. H. van Berkel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Styrene Oxide ◽  
Functional Expression ◽  
Rhodococcus Opacus ◽  
Reading Frame ◽  
Arthrobacter Aurescens ◽  
C Terminus ◽  
Two Component ◽  
Rhodococcus Opacus 1Cp

ABSTRACT 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.

scholarly journals Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in Rhodococcus opacus 1cp

2000 ◽  
Vol 66 (5) ◽  
pp. 2148-2153 ◽  
Author(s):  
Zoya I. Finkelstein ◽  
Boris P. Baskunov ◽  
Marelle G. Boersma ◽  
Jacques Vervoort ◽  
Eugene L. Golovlev ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Mass Spectrum ◽  
Magnetic Resonance ◽  
Thin Layer ◽  
Chemical Shifts ◽  
Rhodococcus Opacus ◽  
Whole Cells ◽  
Mass Spectrum Analysis ◽  
Rhodococcus Opacus 1Cp ◽  
Layer Chromatography

ABSTRACT The transformation of monofluorophenols by whole cells ofRhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and 19F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The 19F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus1cp.

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