The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.     

Mycobacterium paraterrae sp. nov. recovered from a clinical specimen: novel chromogenic slow growing mycobacteria related to Mycobacterium terrae complex

A previously unidentified, slowly growing scotochromogenic Mycobacterium was isolated from a Korean patient with symptomatic pulmonary infection. Phenotypically, this strain was generally similar to Mycobacterium terrae complex strains, however it uniquely produced orange pigmentation. Unique mycolic acid profiles and phylogenetic analyses based on three alternative chronometer molecules, 16S rRNA gene, hsp65 and rpoB , confirmed the taxonomic status of this strain as a novel species. These results support that this strain represents a novel Mycobacterium species. The name Mycobacterium paraterrae sp. nov. is proposed. The type strain is 05-2522 (= DSM 45127 = KCTC 19556).     

1, 2-propanediol from the Pacific Ocean fish Ctenochaetus striatus

(±) 1, 2-propanediol has been isolated from the fish Ctenochaetus striatus. Obtained a first time as the free alcohol with a high yield, it has been found in smaller amount and esterified, in specimens of a different location. The possible origin of this compound is discussed.     

The catechol 2,3-dioxygenase gene and toluene monooxygenase genes from Burkholderia sp. AA1, an isolate capable of degrading aliphatic hydrocarbons and toluene

Burkholderia sp. AA1 isolated from a diesel fuel-contaminated site degraded toluene, as well as a wide range of alkanes from decane (C₈) to pentacosane (C₂₅) as sole carbon and energy sources. This strain also utilized m-toluate, p-toluate, o-toluate, and m-cresol as sole carbon and energy sources. Toluene- and toluate-grown cells showed catechol 2,3-dioxygenase activity and indole oxidation activity that is exhibited by some toluene oxygenation enzymes. The catechol 2,3-dioxygenase gene (catB) was cloned and sequenced. Its deduced amino acid sequence is analogous to the extradiol dioxygenases cloned from a variety of microorganisms. A DNA fragment containing the genes for the indole oxidation activity was cloned and sequenced. A seven-gene cluster designated as tbhABCDEFG was identified. Significant similarities were found with multicomponent monooxygenase systems for toluene, benzene and phenol from different bacterial strains. Journal of Industrial Microbiology & Biotechnology (2000) 25, 127–131. Received 28 July 1999/ Accepted in revised form 28 June 2000     

A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli

We cloned a gene from Methylophaga sp. strain SK1. This gene was responsible for producing, the blue pigment, indigo. The complete open reading frame was 1371 bp long, which encodes a protein of 456 amino acids. The molecular mass of the encoded protein was 105 kDa, consisting of homodimer of 54 kDa with an isoelectric point of 5.14. The deduced amino acid sequence from the gene showed approximately 30% identities with flavin-containing monooxygenases (FMOs) of human (FMO1-FMO5), Arabidopsis, and yeast. It contained three characteristic sequence motifs of FMOs: FAD binding domain, FMO-identifying sequence motif, and NADPH binding domain. In addition, the biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, we assigned the enzyme to be a bacterial FMO. The recombinant Escherichia coli expressing the bacterial FMO produced up to 160 mg of indigo per liter in the tryptophan medium after 12h cultivation. This suggests that the recombinant strain has a potential to be applied in microbial indigo production.     

Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver

The FAD-containing monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these two sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Km- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Km-values for all of the amines and disulfides tested. Mouse FMO had Km-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax-values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Km = 0.97 and 1.1 microM, respectively) and for NADH (Km = 48 and 73 microM, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO but not with mouse liver FMO, indicating structural differences between these two enzymes. This antibody inhibited pig FMO activity up to 60%.     

Cloning and characterization of a cyclohexanone monooxygenase gene from Arthrobacter sp. L661

Cyclohexanone monooxygenase (CHMO), a type of Baeyer-Villiger oxidation, catalyzes the oxidation of cyclohexanone into ɛ-caprolactone, which has been utilized as a building block in organic synthesis. A bacterium that is capable of growth on cyclohexanone as a sole carbon source was recently isolated and was identified as Arthrobacter sp. L661. The strain is believed to harbor a CHMO gene (chnB), considering the high degradablity of cyclohexanone. In order to characterize the CHMO, a chnB gene was cloned from Arthrobacter sp. L661. The deduced amino acids of the chnB gene evidenced the highest degree of homology (90% identity) with the CHMO of Arthrobacter sp. BP2 (accession no. AY123972). The CHMO of L661 was shown to be functionally expressed in Escherichia coli cells, purified via affinity chromatography, and characterized. The specific activity of the purified enzyme was 24.75 μmol/min/mg protein. The optimum pH was 7.0 and the enzyme maintained over 70% of its activity for up to 24 h in a pH range of 6.0 to 8.0 at 4°C. The CHMO of L661 readily oxidized cyclobutanone and cyclopentanone whereas less activity was detected with those of Arthrobacter sp. BP2, Rhodococcus sp. Phi1, and Rhodococcus sp. Phi2, thereby suggesting that the CHMO of L661 evidenced the different substrate specificities compared with other CHMOs. These results can provide us with useful information for the development of biocatalysts applicable to commercial organic syntheses, especially because only a few CHMO genes have been identified thus far.     

Comparative analysis of gene sequences encoding ammonia monooxygenase of Nitrosospira sp. AHB1 and Nitrosolobus multiformis C-71

Abstract DNA encoding ammonia monooxygenase from two phylogenetically related autotrophic nitrifying bacteria, Nitrosospira sp. AHB1 and Nitrosolobus multiformis C-71, was amplified by PCR. The resulting products were cloned into the vector pCR-Script. A continuous region of DNA of about 1.5 kb for strain AHB1 and 1.24 kb for N. multiformis C-71 was analysed. These comprised the major part of the gene amoA encoding the active site polypeptide and, directly downstream, the 5' portion of the amoB gene. The identity values for these sequences at the amino acid level were 93.0% for amoA and 96.1% for amoB . The corresponding values for the nucleic acid sequences were 86.7% and 88.8%, respectively. The identity of the 16S rRNA gene of strain AHB1 to that of N. multiformis C-71 was at least 98.5%. The different degree of sequence conservation between the 16S rDNA and the genes encoding for ammonia monooxygenase facilitates the application of the latter as a molecular tool for a fine-scale differentiation of autotrophic nitrifying bacteria, at the species or strain level, in both environmental and cultivation studies.     

Detection and purification of a catalase-peroxidase from Mycobacterium sp. Pyr-1

A catalase-peroxidase from Mycobacterium sp. Pyr-1, a strain capable of growth on pyrene, was purified to homogeneity by anion exchange and hydroxyapatite column chromatography. The enzyme, like the M. tuberculosis T-catalase, reduced nitroblue tetrazolium in the presence of isoniazid (INH) and H2O2. It also oxidized 3,3',5,5'-tetramethylbenzidine and other substrates of the catalase-peroxidase of M. tuberculosis in the presence of either tert-butyl hydroperoxide or H2O2. It had a UV/ visible absorption spectrum (Soret peak at 406 nm) similar to that of the catalase-peroxidase of M. tuberculosis (Soret peak at 408 nm) and identical to that of the catalase-peroxidase of M. smegmatis. After electrophoresis on non-denaturing gels the enzyme showed one single protein band with both catalase and peroxidase activity, which were lost after electrophoresis on SDS-PAGE. The enzyme was inhibited by sodium azide, glutathione, 2-mercaptoethanol, and isoniazid, but not by isonicotinic acid. The optimum enzyme activity was obtained at pH 4.5 and at 25 degrees C.     

Mycobacterium massiliense is differentiated from Mycobacterium abscessus and Mycobacterium bolletii by erythromycin ribosome methyltransferase gene (erm) and clarithromycin susceptibility patterns

Erythromycin ribosome methyltransferase gene (erm) sequences of Mycobacterium massiliense and Mycobacterium bolletii isolates were newly investigated. Forty nine strains of M. massiliense that were analyzed in the present study had a deleted erm(41). Due to a frame‐shift mutation, large deletion, and truncated C‐terminal region, the Erm(41) of M. massiliense had only 81 amino acids encoded by 246 nucleotides. Corresponding to these findings, most of the M. massiliense isolates (89.8%) were markedly clarithromycin susceptible, but resistant strains invariably had a point mutation at the adenine (A₂₀₅₈ or A₂₀₅₉) in the peptidyltransferase region of the 23S rRNA gene, which is quite different from Mycobacterium abscessus and M. bolletii. In addition, erm(41) sequences of M. massiliense were more conserved than those of M. abscessus and M. bolletii. The results of species identification using erm(41) showed concordant results with those of multi‐locus sequence analysis (rpoB, hsp65, sodA and 16S‐23S ITS) where there were originally inconsistent results between rpoB and hsp65 sequence analysis in previous research. Therefore, erm(41) PCR that was used in the present study can be efficiently used to simply differentiate M. massiliense from M. abscessus and M. bolletii.     

Isolation and identification of a plant growth promotive substance from mixture of essential plant oils

The PCS (commercial products by Field Science Co, Japan, used for air fresheners) was analyzed for the presence of bioactive constituents and their role as root growth promoters. Chromatographic separation of the methanolic solution of PCS resulted in the isolation of an promoting active substance, which was identified using GC-mass spectrometry and NMR spectroscopy as 1,2-propanediol (CH₃CH(OH)CH₂OH). Lettuce seedling growth bioassay as test plant revealed that 1,2-propanediol can act as potent root growth promoter; enhancing the growth of lettuce seedling radicle at a concentration 0.01 ppm. The concentration of 1,2-propanediol in PCS mixture was estimated as 4 g/l. These studies suggest that 1,2-propanediol might play an important role in the plant growth promoting activity of PCS.     

Purification and crystallization of the hydroxylase component of the methanesulfonate monooxygenase from Methylosulfonomonas methylovora strain M2

The aim of the work described in this paper was two-fold: (1) the purification of the hydroxylase component of the MSAMO to electrophoretic homogeneity using a four-step chromatographic strategy and (2) the crystallization of the two-component hydroxylase of the MSAMO in order to enhance our understanding of the precise three-dimensional structure of the MSAMO, thus yielding an insight into the nature of the active site of this enzyme. Optimised crystallization conditions were identified allowing growth of crystals of the hydroxylase component of the MSAMO within five days. Crystals exhibited a brown colour suggesting the presence on an intact Rieske-iron sulfur centre and diffracted to 7.0A when a few degrees of data were evaluated on a beam line X11.     

Identification and Characterization of a Mycobacterial (2R,3R)-2,3-Butanediol Dehydrogenase

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD⁺-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.     

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.     

Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M

The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out.     

Heterologous expression of alkene monooxygenase components from Xanthobacter autotrophicus Py2 and reconstitution of the active complex

The coupling protein and ferredoxin from Xanthobacter autotrophicus Py2 alkene monooxygenase (Xamo) have been functionally expressed in both N-terminal affinity tagged fusion and native forms in Escherichia coli. However, attempts to express the NADH-oxidoreductase and oxygenase, always resulted in the production of inactive, insoluble proteins. Nevertheless, the recombinant reductase from the toluene 4-monooxygenase of Pseudomonas mendocina KR1 was found to functionally complement the Xamo system. In vitro reconstitution, using the recombinant coupling protein and other components purified from the wild type, showed that steady-state epoxidation rate and coupling efficiency were dependent on the relative concentration of Xamo components in the reaction. The optimal molar stoichiometric ratio of Xamo components was determined to be approximately 1:0.25-0.3:2:2 (oxygenase hexamer:reductase:ferredoxin:coupling protein), suggesting the formation of an efficient catalytic complex at the minimal stoichiometric ratio to saturate the probable two-fold symmetry binding sites on the oxygenase.     

Complete genome sequence of Mycobacterium sp. strain (Spyr1) and reclassification to Mycobacterium gilvum Spyr1

Mycobacterium sp.Spyr1 is a newly isolated strain that occurs in a creosote contaminated site in Greece. It was isolated by an enrichment method using pyrene as sole carbon and energy source and is capable of degrading a wide range of PAH substrates including pyrene, fluoranthene, fluorene, anthracene and acenapthene. Here we describe the genomic features of this organism, together with the complete sequence and annotation. The genome consists of a 5,547,747 bp chromosome and two plasmids, a larger and a smaller one with sizes of 211,864 and 23,681 bp, respectively. In total, 5,588 genes were predicted and annotated.     

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å), and its complex with hydrolysis intermediate 5′-pApA (2.35 Å). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.     

Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat.

Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatography of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1alpha. The recombinant clones having higher potentiality to degrade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.