Heterologous expression of alkene monooxygenase from Rhodococcus rhodochrous B-276.

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 is a three-component enzyme system encoded by the four-gene operon amoABCD. AMO catalyses the stereoselective epoxygenation of aliphatic alkenes, yielding primarily R enantiomers. The presumed site of alkene oxygenation is a dinuclear iron centre similar to that in the soluble methane monooxygenases of methanotrophic bacteria, to which AMO exhibits a significant degree of amino acid sequence identity. The AMO complex was not expressed in Escherichia coli, at least partly because that host did not produce all of the AMO polypeptides. Expression of AMO was achieved in Streptomyces lividans by cloning the AMO genes into the thiostrepton-inducible expression plasmid pIJ6021. No background of AMO activity was detected in S. lividans cells without amoABCD and expression of AMO activity, at a level comparable to that from wild-type R. rhodochrous B-276, coincided with appearance of the AMO subunits. Recombinant AMO activity in cell-free extracts of S. lividans was stimulated by the addition of NADH and produced R-epoxypropane with comparable enantiomeric excess to AMO purified from the original organism. Although the whole AMO complex could not be expressed in E. coli, the functional coupling protein (AmoB) and reductase (AmoD) were expressed individually in E. coli as fusions with glutathione S-transferase. The expression systems described here now allow structure/function studies on AMO to be carried out by site-directed mutagenesis.     

Cloning,sequencing and functional expression of a DNA encoding pig cytosolic malate dehydrogenase: purification and characterization of the recombinant enzyme

Using the polymerase chain reaction, DNA encoding cytosolic malate dehydrogenase (cMDH) has been cloned from a pig heart cDNA library. Large amounts of the enzyme (30 mg per litre of original culture) have been produced in Escherichia coli using an inducible expression vector (pKK223-3) in which the 5′-non-coding region of the gene was replaced with the tac promoter. The complete nucleotide sequence of the DNA is reported for the first time. The recombinant cMDH purified was shown to be identical to the native enzyme according to: chromatographic behaviour, isoelectric point, N-terminal amino acid sequence, and physicochemical and catalytic properties.     

Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization

Nitrilase was purified from an extract of isovaleronitrile-induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45 degrees C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, L-cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3-chlorobenzonitrile, 4-tolunitrile, 2-furonitrile and 2-thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.     

Cloning,sequencing, and characterization of the hexahydro-1,3,5-Trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.     

Isolation and primary characterization of an amidase from Rhodococcus rhodochrous

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.     

Hydroaromatic metabolism in Rhodococcus rhodochrous: purification and characterisation of its NAD-dependent quinate dehydrogenase

Quinate grown cells of Rhodococcus rhodochrous N75 metabolized both quinate and shikimate via protocatechuate to succinate and acetyl CoA. The initial enzyme of the hydroaromatic pathway, quinate dehydrogenase was purified 188-fold to electrophoretic homogeneity. The enzyme is a monomer with a native relative molecular mass of 44,000 and is NAD-dependent. The enzyme is highly stereospecific with regard to hydroaromatic substrates, oxidising only the axial hydroxyl group at C-3 of (-)-isomers of quinate, shikimate, dihydroshikimate and t-3,t-4-dihydroxycyclohexane-c-1-carboxylate, but shows activity with several NAD analogues.     

The molecular cloning and sequencing of the nitrilase gene of Rhodococcus rhodochrous PA-34

The nitrilase of Rhodococcus rhodochrous PA-34 catalyzes the production of optically active amino acids from aminonitriles. The amino acid sequence of the NH₂ terminus of the purified nitrilase was determined for the preparation of a synthetic oligonucleotide as a southern hybridization probe. A 9.5-kbp Pst I-fragement, which hybridized with the oligonucleotide probe, was isolated from R. rhodochrous PA-34 genomic libraries constructed in pUC 19. Nucleotide sequence analysis revealed that the nitrilase gene codes for a putative polypeptide of 380 amino acids which correspond to a relative molecular weight of 41, 723.     

A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.     

Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme.

The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a Km of 4.8 +/- 0.9 microM, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis.     

Functional expression, purification, and characterization of the recombinant Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700

For the investigation of the NADPH-dependent Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700, the encoding gene mekA with a C-terminal strep-tag was cloned and expressed under the control of a l-rhamnose inducible promoter from Escherichia coli. The mekA gene was found by analyzing the methylethylketone (MEK) degradation pathway by Onaca et al. J Bacteriol 189:3759–3767, 2007. Sequence analysis of the corresponding protein, which catalyzes the Baeyer-Villiger oxidation of MEK to ethyl acetate, showed two binding sites (Rossman-fold motifs) for cofactors NAD(P)H and FAD. Although expression of mekA resulted in large amounts of inclusion bodies compared to soluble protein, high amounts of purified and active MekA were obtained by affinity chromatography. The substrate spectrum of MekA was investigated with purified enzyme and whole cells using a variety of aliphatic, aromatic, and cyclic ketones including four chiral substrates. The specific activity of MekA with MEK as substrate was determined to be 1.1 U/mg protein. K _M values were determined for MEK and the cofactors NADPH and NADH to be 6, 11, and 29 μM, respectively.     

Cloning,sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme

The bovine pancreatic (bp-) DNase I gene has been cloned from bp-cDNA and expressed in E. coli. A polynucleotide sequence of 1295 base pairs was deduced from clones of the cDNA. The sequence showed an open reading frame which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bp-DNase I. An expression plasmid was constructed by inserting into the vector pET-15b, a cDNA fragment coding for bp-DNase I ligated with a hexanucleotide coding for Met–Ala at the 5′-end. The plasmid was transformed into E. coli strain DH5α and the active recombinant bovine (rb-) DNase I was produced after induction of protein synthesis. From the induced culture medium, rb-DNase I was purified by chromatography on a Mono Q column. The purified rb-DNase I showed a molecular mass of 29 kDa and had the same specific activity as bp-DNase I. The NH₂-terminus of rb-DNase I was Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bp-DNase I, Asn was not glycosylated.     

Spectroscopic characterization of a newly isolated cytochrome P450 from Rhodococcus rhodochrous.

Cytochrome P450 (P450) from Rhodococcus rhodochrous have been characterized through circular dichroism and nuclear magnetic resonance (NMR) spectroscopy, both in the substrate-free and substrate-bound forms. The data are compared with those of P450cam and indicate a close similarity of the structure of the active site in the two proteins. The substrate-free species contains low-spin iron(III), while the 2-ethoxyphenol bound species contains high-spin iron(III). The substrate is in slow exchange on the NMR time scale. The binding of CN- has been investigated and the final adduct characterized through NMR spectra. Nuclear relaxation times of the isotropically shifted signals turn out to be shorter than in other heme proteins, both in the high- and in the low-spin species. This is the result of longer electron relaxation times in P450s than in peroxidases and metmyoglobin. This property, as well as the electron paramagnetic resonance (EPR) spectrum of the substrate-free form, are discussed in terms of the presence of the cysteine as the fifth ligand of the iron ion instead of a histidine as it occurs in peroxidases and myoglobin.     

Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme.

NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism.     

Purification and characterization of rhodobactin: a mixed ligand siderophore from Rhodococcus rhodochrous strain OFS

The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D ¹H, ¹³C and ¹⁵N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 μM to 2.0 μM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

The DNA untwisting enzyme from Saccharomyces cerevisiae. Partial purification and characterization.

The DNA untwisting enzyme has been partially purified from Saccharomyces cerevisiae. The enzyme exhibits a pH optimum of 7.3 to 7.6 in phosphate buffer, appears to require 0.15 M KCl for activity as determined by a DNA filter-binding assay, and is inhibited by N-ethylmaleimide. Like the untwisting enzymes from other eucaryotic cells, it can remove both positive and negative superhelical turns. A DNA molecule containing a single strand break was shown to be an intermediate in the untwisting reaction. Thermal stabilities of the enzyme from selected conditional lethal mutants defective in DNA synthesis have been examined and were found to be indistinguishable from the wild type enzyme.     

Characterization of the genome of the Rhodococcus rhodochrous bacteriophage NJL.

Temperate bacteriophage NJL of Rhodococcus rhodochrous has a 49-kb linear double-stranded DNA with cohesive ends (cos). NJL DNA has unique target sites for HindIII and SspI, two target sites each for NheI and ScaI, and no cleavage site for AxyI, DraI, EcoRI, SacI, and SphI. The single-stranded regions of cos ends were ligated to each other with T4 DNA ligase, removed with mung bean nuclease, or blunted with the Klenow large fragment of DNA polymerase I; then the sequences of the cos ends were determined. Comparison of these sequences revealed that the single-stranded regions are complementary and 18 bases long and protrude at the 3' ends; they have the following sequences: 5'-TTGGCACCGTGGGAGGAG-3' and 3'-AACCGTGGCAC CCTCCTC-5'. A physical map of NJL was constructed by a cos mapping method based on information about the structure of the cohesive ends and multiple digestions with restriction endonucleases.     

Over-expression, purification, and characterization of recombinant NAD-malic enzyme from Escherichia coli K12

NAD(+)-dependent malic enzyme (NAD-ME) gene from Escherichia coli K12 was inserted into an expression vector pET24b(+) and transformed into E. coli BL21 (DE3). Recombinant NAD-ME was expressed upon IPTG induction, purified with affinity chromatography, and biochemically characterized. The results showed that recombinant NAD-ME could be produced mainly in a soluble form. The monomeric molecular weight of recombinant NAD-ME was about 65 kDa, whereas monomer, homotetramer, and homooctamer were formed in solution as revealed by nondenaturing polyacrylamide gel electrophoresis analysis. Finally, the K(m) values of NAD-ME for L-malate and NAD were determined as 0.420+/-0.174 and 0.097+/-0.038 mM, respectively, at pH 7.2. By using this over-expression and purification system, recombinant E. coli K12 NAD-ME can now be obtained in large quantity necessary for further biochemical characterization and applications.     

Catabolic alanine racemase from Salmonella typhimurium: DNA sequence, enzyme purification, and characterization

The alanine racemase encoded by the Salmonella typhimurium dadB gene was purified to 90% homogeneity from an overproducing strain. At 37 degrees C the enzyme has a specific activity of 1400 units/mg (V max, L- to D-alanine). Active enzyme molecules are monomers of Mr 39 000 with one molecule of pyridoxal 5'-phosphate bound per subunit. The Km's for L- and D-alanine are 8.2 and 2.1 mM, respectively. Measurement of turnover numbers yielded the expected Keq value of 1.0. Determination of 22 of the 25 N-terminal amino acid residues of the purified polypeptide allowed localization of cloned DNA encoding the structural gene. Sequencing of subcloned DNA revealed that the dadB gene encodes a polypeptide of 356 amino acids whose calculated molecular weight (apoenzyme) was 39 044.