Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP.

The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.     

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

3-Ketosteroid Δ¹-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ¹-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ¹-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr¹¹⁹, Tyr³¹⁸, Tyr⁴⁸⁷, and Gly⁴⁹¹. Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr³¹⁸, respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr⁴⁸⁷ and the backbone amide of Gly⁴⁹¹. Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr⁴⁸⁷ and Gly⁴⁹¹ work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr¹¹⁹, the general base Tyr³¹⁸ abstracts the axial β-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion.     

Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis.

Three copies of the IS21-related transposable element IS1415 were identified in Rhodococcus erythropolis NI86/21. Adjacent to one of the IS1415 copies, a 47-bp sequence nearly identical to the conserved 5' end of integrons was found. Accurate transposition of IS1415 carrying a chloramphenicol resistance gene (Tn5561) was demonstrated following delivery from a suicide vector to R. erythropolis SQ1.     

Isolation and Characterization of Carbendazim-degrading Rhodococcus erythropolis djl-11

Carbendazim (methyl 1H-benzimidazol-2-yl carbamate) is one of the most widely used fungicides in agriculture worldwide, but has been reported to have adverse effects on animal health and ecosystem function. A highly efficient carbendazim-degrading bacterium (strain dj1-11) was isolated from carbendazim-contaminated soil samples via enrichment culture. Strain dj1-11 was identified as Rhodococcus erythropolis based on morphological, physiological and biochemical characters, including sequence analysis of the 16S rRNA gene. In vitro degradation of carbendazim (1000 mg·L⁻¹) by dj1-11 in minimal salts medium (MSM) was highly efficient, and with an average degradation rate of 333.33 mg·L⁻¹·d⁻¹ at 28°C. The optimal temperature range for carbendazim degradation by dj1-11 in MSM was 25–30°C. Whilst strain dj1-11 was capable of metabolizing cabendazim as the sole source of carbon and nitrogen, degradation was significantly (P<0.05) increased by addition of 12.5 mM NH₄NO₃. Changes in MSM pH (4–9), substitution of NH₄NO₃ with organic substrates as N and C sources or replacing Mg²⁺ with Mn²⁺, Zn²⁺ or Fe²⁺ did not significantly affect carbendazim degradation by dj1-11. During the degradation process, liquid chromatography-mass spectrometry (LC-MS) detected the metabolites 2-aminobenzimidazole and 2-hydroxybenzimidazole. A putative carbendazim-hydrolyzing esterase gene was cloned from chromosomal DNA of djl-11 and showed 99% sequence homology to the mheI carbendazim-hydrolyzing esterase gene from Nocardioides sp. SG-4G.     

Characterization of LtsA from Rhodococcus erythropolis, an enzyme with glutamine amidotransferase activity

The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ltsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ltsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ltsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH(4)Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH(4)Cl, indicating that NH(3) can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.     

Chemical and Physical Characterization of Interfacial-Active Lipids from Rhodococcus erythropolis Grown on n-Alkanes

Lipophilic compounds of the culture suspension containing Rhodococcus erythropolis DSM43215 had surfactant properties when the bacteria were cultivated with n-alkanes as the sole carbon source. Thirteen main components from a dichloromethane-methanol extract of the R. erythropolis cultures were isolated and characterized to specify quantitatively their surfactant properties, e.g., minimum surface and interfacial tensions and critical micelle concentrations. The interfacial activity of the organic extract was dominated by α,α-trehalose-6,6′-dicorynomycolates which reduced interfacial tension from 44 to 18 mN/m. Phosphatidylethanolamines which were also present in the organic extract reduced the interfacial tension below 1 mN/m. The trehalose corynomycolates had extremely low critical micelle concentrations in high-salinity solutions, and the interfacial properties were stabile in solutions with a wide range of pH and ionic strength.     

Aryl acylamidase from Rhodococcus erythropolis NCIB 12273

Summary A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around pH 8.0 and had a K_m for acetaminophen of 0.11 mM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen. Offprint requests to: P. A. Vaughan     

Purification and Characterization of Lipid Bioflocculant Produced by Rhodococcus erythropolis

The bioflocculant produced by Rhodococcus erythropolis S-1 was found to exist as huge assemblies, the molecular mass of which is over one million daltons, composed of many polypeptides and lipids in aqueous solution. We have isolated and purified this lipid bioflocculant by ultracentrifugation, extracting with 90% acetone, and two successive silica gel chromatographies from the culture broth. It was homogeneous on silica gel thin-layer chromatography. ¹H-NMR and HPLC studies showed that it was a kind of glycolipid that contained a C₁₆ methylene chain on the average and glucose in its chemical structure. The flocculating activity against kaolin clay suspension was dependent on the Ca²⁺ concentration.     

Targeted Disruption of the kstD Gene Encoding a 3-Ketosteroid Δ1-Dehydrogenase Isoenzyme of Rhodococcus erythropolis Strain SQ1

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Δ¹-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Δ¹-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Δ¹-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9α-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Δ¹-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Δ¹-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.     

Assimilation of Propane and Characterization of Propane Monooxygenase from Rhodococcus erythropolis3/89

The ability of propane-assimilating microorganisms of the genus Rhodococcusto utilize metabolites of the terminal and subterminal pathways of propane oxidation was studied. Propane monooxygenase of Rhodococcus erythropolis3/89 was shown to be an inducible enzyme catalyzing epoxidation and hydroxylation of organic compounds. The optimum conditions for the epoxidation of gaseous and liquid alkenes and the hydroxylation of aromatic carbohydrates were found.     

Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Delta(1)-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Delta(1)-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Delta(1)-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9alpha-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Delta(1)-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Delta(1)-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.     

Dienelactone hydrolase from Rhodococcus erythropolis 1 CP: purification and properties

Dienelactone hydrolases (EC 3.1.1.45) have been shown to play an indispensable role in the degradation of chloroaromatic compounds via ortho-cleavage of chlorocatechols. We report on the purification of dienelactone hydrolase of the chlorophenol-utilizing strain Rhodococcus erythropolis 1CP to apparent homogeneity. Dienelactone hydrolase differed fron the corresponding enzymes of other chloroaromatic compound-catabolizing strains in being restricted to substrates with a cis-dienelactone structure. From the cis-dienelactone-hydrolyzing enzyme of a 4-fluorobenzoate-utilizing Burkholderia (Pseudomonas) cepacia strain, it differed considerably in properties such as pH optimum of activity, inhibition by p-chloromercuribenzoate, and amino acid composition. Thus, there is not necessarily a close relationship between substrate specificity and other properties of dienelactone hydrolases.     

The remarkable Rhodococcus erythropolis

Rhodococcus erythropolis cells contain a large set of enzymes that allow them to carry out an enormous number of bioconversions and degradations. Oxidations, dehydrogenations, epoxidations, hydrolysis, hydroxylations, dehalogenations and desulfurisations have been reported to be performed by R. erythropolis cells or enzymes. This large array of enzymes fully justifies the prospective application of this bacterium in biotechnology.     

Purification and Properties of Gentisate 1,2-Dioxygenase from Rhodococcus erythropolis S-1

Gentisate 1,2-dioxygenase, which participates in salicylate and m-hydroxybenzoate metabolism, was purified from cell-free extracts of Rhodococcus erythropolis S-1, a Gram-positive bacterium. The purified enzyme gave a single band on native PAGE and SDS–PAGE. The molecular mass of the enzyme was estimated to be 328 kDa. The structure of the enzyme appears to be an octamer of identical subunits. The enzyme from this bacterium was similar in general enzymatic properties to a gentisate 1,2-dioxygenase from a Gram-negative bacterium except for molecular mass and structure.     

Selective Desulfurization of Dibenzothiophene by Rhodococcus erythropolis D-1

A dibenzothiophene (DBT)-degrading bacterium, Rhodococcus erythropolis D-1, which utilized DBT as a sole source of sulfur, was isolated from soil. DBT was metabolized to 2-hydroxybiphenyl (2-HBP) by the strain, and 2-HBP was almost stoichiometrically accumulated as the dead-end metabolite of DBT degradation. DBT degradation by this strain was shown to proceed as DBT → DBT sulfone → 2-HBP. DBT at an initial concentration of 0.125 mM was completely degraded within 2 days of cultivation. DBT at up to 2.2 mM was rapidly degraded by resting cells within only 150 min. It was thought this strain had a higher DBT-desulfurizing ability than other microorganisms reported previously.     

Isobutylidenediurea degradation by Rhodococcus erythropolis

A new enzyme (isobutylidenediurea amidinohydrolase) catalyzing the hydrolysis of isobutylidenediurea (a condensation product of urea and isobutyraldehyde widely used as a slow-release nitrogeneous fertilizer) was characterized from a strain of Rhodococcus erythropolis. The enzyme was purified 1250-fold to apparent homogeneity and shown to hydrolyze the fertilizer to urea and isobutyraldehyde at a molar ratio of 2 : 1. No activity was observed with ureido- or other structurally related compounds. Its molecular mass was determined by native polyacrylamide gelelectrophoresis and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry to be 15 kDa (±2 kDa) and 16.4 kDa, respectively. Growth of the bacterium in the presence of isobutylidenediurea led to an increased expression of the constitutively synthetized enzyme.     

Purification and Properties of 3 Types of Monohydroxybenzoate Oxygenase from Rhodococcus erythropolis S-1

Three types of monohydroxybenzoate oxygenase, salicylate 5-oxygenase (SAL5O) forming gentisate from salicylate, m-hydroxybenzoate 6-oxygenase (MHB6O) forming gentisate from m-hydroxybenzoate, and p-hydroxybenzoate 3-oxygenase (PHB3O) forming protocatechuate from p-hydroxybenzoate, were purified from a cell-free extract of Rhodococcus erythropolis S-1, a Gram-positive bacterium. Each purified enzyme was homogenous on native PAGE. Each enzyme was a tetramer having identical subunits, a flavoporotein containing FAD, and a NADH-dependent monooxygenase. The three enzymes were much alike in general enzymatic properties, but very different in substrate specificity.     

Characterization of muconate and chloromuconate cycloisomerase from Rhodococcus erythropolis 1CP: indications for functionally convergent evolution among bacterial cycloisomerases.

Muconate cycloisomerase (EC 5.5.1.1) and chloromuconate cycloisomerase (EC 5.5.1.7) were purified from extracts of Rhodococcus erythropolis 1CP cells grown with benzoate or 4-chlorophenol, respectively. Both enzymes discriminated between the two possible directions of 2-chloro-cis, cis-muconate cycloisomerization and converted this substrate to 5-chloromuconolactone as the only product. In contrast to chloromuconate cycloisomerases of gram-negative bacteria, the corresponding R. erythropolis enzyme is unable to catalyze elimination of chloride from (+)-5-chloromuconolactone. Moreover, in being unable to convert (+)-2-chloromuconolactone, the two cycloisomerases of R. erythropolis 1CP differ significantly from the known muconate and chloromuconate cycloisomerases of gram-negative strains. The catalytic properties indicate that efficient cycloisomerization of 3-chloro- and 2,4-dichloro-cis,cis-muconate might have evolved independently among gram-positive and gram-negative bacteria.