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Jan R Andreesen

Publications and source records attributed to Jan R Andreesen.

16 recordsLinked to original sources
PubMed2006

Peroxidase activity of selenoprotein GrdB of glycine reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum.

The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific "protein B"-complex of glycine reductase. The "protein B"-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)(-1) with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)(-1) in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep-tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.

Amino Acid Oxidoreductases↗
PubMed2006

Pseudonocardia tetrahydrofuranoxydans sp. nov.

A Gram-positive, rod-shaped, non-endospore-forming but mycelium-forming actinobacterium (strain K1(T)) was isolated from an enrichment culture containing tetrahydrofuran (THF) as the sole source of carbon. On the basis of its G+C content (71.3 mol%) and of 16S rRNA gene sequence similarity studies, strain K1(T) was shown to belong to the family Pseudonocardiaceae, most closely related to Pseudonocardia hydrocarbonoxydans (99.3 %), P. benzenivorans (98.8 %) and P. sulfidoxydans (98.3 %). The 16S rRNA gene sequence similarity to other Pseudonocardia species was less than 97 %. Chemotaxonomic data [major menaquinone MK-8(H(4)); major fatty acids C(16 : 0) iso, C(15 : 0) iso and C(17 : 1)omega6c] supported the affiliation of strain K1(T) to the genus Pseudonocardia. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain K1(T) from the three species P. benzenivorans, P. sulfidoxydans and P. hydrocarbonoxydans, although all four organisms utilized THF. Strain K1(T) represents a novel species, for which the name Pseudonocardia tetrahydrofuranoxydans sp. nov. is proposed, with the type strain K1(T) (=DSM 44239(T)=CIP 109050(T)).

Actinomycetales↗
PubMed2005

Analysis of the nearly identical morpholine monooxygenase-encoding mor genes from different Mycobacterium strains and characterization of the specific NADH : ferredoxin oxidoreductase of this cytochrome P450 system.

Cloning and sequencing of the morABC operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by Mycobacterium sp. strain HE5. The cytochrome P450 (P450(mor)) and the Fe(3)S(4) ferredoxin (Fd(mor)), encoded by morA and morB, respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450(mor) system. Analysis of the mor operon has now revealed the gene morC, encoding the ferredoxin reductase of this morpholine monooxygenase. The genes morA, morB and morC were identical to the corresponding genes from Mycobacterium sp. strain RP1. Almost identical mor genes in Mycobacterium chlorophenolicum PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in Mycobacterium sp. strain HE5. Analysis of the downstream sequences of morC revealed differences in this gene region between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 on the one hand, and M. chlorophenolicum on the other hand, indicating insertions or deletions after recombination. Downstream of the mor genes, the gene orf1', encoding a putative glutamine synthetase, was identified in all studied strains. The gene morC of Mycobacterium sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR(mor) was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The K(m) values of FdR(mor) for the substrate NADH (37.7 +/- 4.1 microM) and the artificial electron acceptors potassium ferricyanide (14.2 +/- 1.1 microM) and cytochrome c (28.0 +/- 3.6 microM) were measured. FdR(mor) was shown to interact functionally with its natural redox partner, the Fe(3)S(4) protein Fd(mor), and with the Fe(2)S(2) protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fd(mor) in activity assays. These results indicated that FdR(mor) can utilize different ferredoxins, but that Fd(mor) requires the specific NADH : ferredoxin oxidoreductase FdR(mor) from the P450(mor) system for efficient catalytic function.

Cytochrome P-450 Enzyme System↗
PubMed2005

Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5.

The P450mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fdmor) and cytochrome P450 (P450mor). In this study, we purified Fdmor and P450mor as recombinant proteins as well as FdRmor, which has been isolated previously. Kinetic investigations of the redox couple FdRmor/Fdmor revealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazolium (NBT) and an absolute requirement for Fdmor in this reaction, compared with the NADH-dependent reduction of cytochrome c. The quite low Km (5.3 +/- 0.3 nm) of FdRmor for Fdmor, measured with NBT as the electron acceptor, indicated high specificity. The addition of sequences providing His-tags to the N- or C-terminus of Fdmor did not significantly alter kinetic parameters, but led to competitive background activities of these fusion proteins. Production of P450mor as an N-terminal His-tag fusion protein enabled the purification of this protein in its spectral active form, which has previously not been possible for wild-type P450mor. The proposed substrates morpholine, piperidine or pyrrolidine failed to produce substrate-binding spectra of P450mor under any conditions. Pyridine, metyrapone and different azole compounds generated type II binding spectra and the Kd values determined for these substances suggested that P450mor might have a preference for more bulky and/or hydrophobic molecules. The purified recombinant proteins FdRmor, Fdmor and P450mor were used to reconstitute the homologous P450-containing mono-oxygenase, which was shown to convert morpholine.

Catalysis↗
PubMed2004

Glycine reductase mechanism.

The ability of some anaerobic bacteria to conserve energy via a soluble substrate level phosphorylation system by reducing glycine to acetyl-phosphate has been an intriguing mechanism for about half a century. The genes implicated in this system have been sequenced and form an operon structure with those of the thioredoxin system. The deduced proteins exhibit high degrees of similarity with glycine reductase from other bacteria. Faster progress in understanding the exact mechanisms is hampered, for example, by some unique reactions involving selenoethers and redox active selenocysteines, which do not allow an easy heterologous formation in Escherichia coli. Further major obstacles are the processing of a substrate-specific pro-protein to a new carbonyl/pyruvoyl group in one of the two peptides formed that stabilize the substrate-binding selenoprotein, which contains an additional rather unstable carbonyl group.

Amino Acid Oxidoreductases↗
PubMed2004

Tungsten-containing aldehyde oxidoreductase of Eubacterium acidaminophilum.

Aldehyde oxidoreductase of Eubacterium acidaminophilum was purified to homogeneity under strict anaerobic conditions using a four-step procedure. The purified enzyme was present as a monomer with an apparent molecular mass of 67 kDa and contained 6.0 +/- 0.1 iron, 1.1 +/- 0.2 tungsten, about 0.6 mol pterin cofactor and zinc, but no molybdenum. The enzyme activity was induced if a molar excess of electron donors, such as serine and/or formate, were supplied in the growth medium compared to readily available electron acceptors such as glycine betaine. Many aldehydes served as good substrates, thus enzyme activity obtained with acetaldehyde, propionaldehyde, butyraldehyde, isovaleraldehyde and benzaldehyde differed by a factor of less than two. Kinetic parameters were determined for all substrates tested. Oligonucleotides deduced from the N-terminal amino acid sequence were used to isolate the encoding aorA gene and adjacent DNA regions. The deduced amino acid sequence of the aldehyde oxidoreductase exhibited high similarities to other tungsten-containing aldehyde oxidoreductases from archaea. Transcription of the aorA gene was monocistronic and started from a sigma 54-dependent promoter. Upstream of aorA, the gene aorR is localized whose product is similar to sigma 54-dependent transcriptional activator proteins and, thus, AorR is probably involved in the regulation of aorA expression.

Aldehyde Oxidoreductases↗
PubMed2003

Identification and characterization of the cytoplasmic tungstate/molybdate-binding protein (Mop) from Eubacterium acidaminophilum.

The mop gene, encoding the molybdate-binding protein from Eubacterium acidaminophilum, was cloned using Clostridium pasteurianum mopI as a probe for heterologous hybridization. mop encodes a 69-amino-acid protein ( M(r) 7,328) with high sequence similarities to members of the molbindin protein family, which have been implicated in molybdenum storage and homeostasis. Northern blot analysis showed three mRNA transcripts (1.0, 1.6, and 2.6 kb) for mop. This result was obtained independent of the availability of tungstate in the growth medium. mop was overexpressed in Escherichia coli as a C-terminal Strep-tag fusion protein. On the basis of gel filtration, the native protein was a homohexamer of 48 kDa. The specificity of oxyanion binding was examined by protein mobility shift assay. Molybdate, tungstate, and chromate strongly changed the mobility of the protein in a native polyacrylamide gel, indicating the binding of these oxyanions to Mop. Other oxyanions, such as sulfate and phosphate, had no effect on Mop mobility. Mutational analysis revealed that the positive charge of the Arg-6, located in the conserved SARN region of Mop, was not directly involved in oxyanion binding.

Amino Acid Sequence↗
PubMed2003

Structural studies on a twin-arginine signal sequence.

Translocation of folded proteins across biological membranes can be mediated by the so-called 'twin-arginine translocation' (Tat) system. To be translocated, Tat substrates require N-terminal signal sequences which usually contain the eponymous twin-arginine motif. Here we report the first structural analysis of a twin-arginine signal sequence, the signal sequence of the high potential iron-sulfur protein from Allochromatium vinosum. Nuclear magnetic resonance (NMR) analyses of amide proton resonances did not indicate a signal sequence structure. Accordingly, data from H/D exchange matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry showed that the amide protons of the signal sequence exchange rapidly, indicating the absence of secondary structure in the signal sequence up to L29. We conclude that the conserved twin-arginine motif does not form a structure by itself or as a result of intramolecular interactions.

Amino Acid Sequence↗
PubMed2003

Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K1.

A gene cluster involved in the utilization of tetrahydrofuran by Pseudonocardia sp. strain K1 was cloned and sequenced. Analysis of a 9.2-kb DNA fragment revealed eight ORFs. The genes designated as thmADBC encode the components of a putative monooxygenase exhibiting a high similarity to different binuclear-iron-containing multicomponent monooxygenases. thmA encodes the derived 545-amino-acid oxygenase alpha-subunit, thmD the 360-amino-acid reductase component, thmB the 346-amino-acid oxygenase beta-subunit, and thmC the 117-amino-acid coupling protein. Upstream of the thm genes, an additional ORF ( sad) was identified coding for a protein with high similarity to various aldehyde dehydrogenases. A succinate semialdehyde dehydrogenase activity was specifically expressed in tetrahydrofuran-grown cells. N-terminal sequence analysis of the purified protein revealed that it is encoded by sad. Northern blot analysis indicated that transcription of the thm genes and sad was specifically induced during growth on tetrahydrofuran. Mono-, di- and polycistronic transcripts of these genes were detected. Primer-extension analysis identified transcriptional start sites 37, 61, and 41 bp upstream of the translation start of sad, thmA, and thmB, respectively. Additional ORFs were identified upstream ( orfY) and downstream ( orfZ and aldH) of the thm genes. Furthermore, the data indicated that the analyzed gene cluster was present as a single copy and located on a plasmid.

Actinomycetales↗
PubMed2003

Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) are among the most notorious environmental pollutants. Some congeners, particularly those with lateral chlorine substitutions at positions 2, 3, 7 and 8, are extremely toxic and carcinogenic to humans. One particularly promising mechanism for the detoxification of PCDDs and PCDFs is microbial reductive dechlorination. So far only a limited number of phylogenetically diverse anaerobic bacteria have been found that couple the reductive dehalogenation of chlorinated compounds--the substitution of a chlorine for a hydrogen atom--to energy conservation and growth in a process called dehalorespiration. Microbial dechlorination of PCDDs occurs in sediments and anaerobic mixed cultures from sediments, but the responsible organisms have not yet been identified or isolated. Here we show the presence of a Dehalococcoides species in four dioxin-dechlorinating enrichment cultures from a freshwater sediment highly contaminated with PCDDs and PCDFs. We also show that the previously described chlorobenzene-dehalorespiring bacterium Dehalococcoides sp. strain CBDB1 (ref. 3) is able to reductively dechlorinate selected dioxin congeners. Reductive dechlorination of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) demonstrates that environmentally significant dioxins are attacked by this bacterium.

Bacteria, Anaerobic↗
PubMed2003

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

Cloning, Molecular↗
PubMed2002

Reclassification of Clostridium hydroxybenzoicum as Sedimentibacter hydroxybenzoicus gen. nov., comb. nov., and description of Sedimentibacter saalensis sp. nov..

Strain ZF2T, isolated from freshwater sediment, is a motile, rod-shaped, gram-positive, endospore-forming, amino acid- and pyruvate-utilizing, anaerobic bacterium. It requires yeast extract for growth. Carbohydrates are not utilized. The optimal temperature and pH for growth are 37 degrees C and 6.8-7.3, respectively. The G+C content of the DNA is 34.0 mol %. A 16S rDNA sequence analysis of strain ZF2T revealed that the highest similarity (94.4%) was shared with Clostridium hydroxybenzoicum JW/Z-1T. Strain ZF2T, however, was not able to carboxylate phenol or to decarboxylate 4-hydroxybenzoate, which are characteristic properties of strain JW/Z-1T. The degree of 16S rDNA relatedness, together with the physiological and chemotaxonomic properties, suggest that strain ZF2T represents a novel species that is clearly distinct from Clostridium hydroxybenzoicum JW/Z-1T. In a phylogenetic dendrogram, both strains form a separate cluster that is peripherally associated with the Peptostreptococcus group (cluster XIII) of the clostridia and the lineage of Helcococcus kunzii. Strains ZF2T and JW/Z-1T show a somewhat deeper branching from the cluster XII clostridia Clostridium purinolyticum and Clostridium acidiurici. The latter strains possessed the closest 16S rDNA similarity (between 88.4 and 90.7%), but were clearly separated by phenotypic markers. Therefore, a new genus, Sedimentibacter gen. nov., is described, comprising Sedimentibacter hydroxybenzoicus gen. nov., comb. nov., as the type species of the genus, with JW/Z-1T (= ATCC 51151T = DSM 7310T) as the type strain, and the novel species Sedimentibacter saalensis sp. nov., with strain ZF2T (= DSM 13558T = ATCC BAA-283T) as the type strain.

Bacterial Typing Techniques↗
PubMed2000

High morpholine degradation rates and formation of cytochrome P450 during growth on different cyclic amines by newly isolated Mycobacterium sp. strain HE5.

Using morpholine as sole source of carbon, nitrogen and energy, strain HE5 (DSM 44238) was isolated from forest soil. The isolated strain was identified as a member of the subgroup of fast-growing Mycobacterium species as revealed by 16S rDNA analysis. An identity of 99.4% was obtained to Mycobacterium gilvum; however, the type strain was unable to utilize morpholine. A maximal growth rate of 0.17 h(-1) was observed at a morpholine concentration of 30 mM, 30 degrees C and pH 7.2. The substrate was tolerated at concentrations up to 100 mM. Besides morpholine, the strain utilized pyrrolidine, piperidine and proposed intermediates in morpholine metabolism such as glycolate, glyoxylate and ethanolamine. Degradation of morpholine, piperidine and pyrrolidine by resting or permeabilized cells was strictly dependent on the presence of oxygen. Addition of the cytochrome-P450-specific inhibitor metyrapone to the growth medium resulted in a significantly decreased growth rate if these cyclic amines were used as a substrate. Carbon monoxide difference spectra of crude extracts from cells grown on these substrates compared to spectra obtained for extracts of succinate-grown cells indicated that cytochrome P450 is specifically expressed during growth on the cyclic amines. These data indicated that a cytochrome-P450-dependent monooxygenase is involved in the degradation of the three cyclic amines.

Amines↗
PubMed1999

Proline biosynthesis from L-ornithine in Clostridium sticklandii: purification of delta1-pyrroline-5-carboxylate reductase, and sequence and expression of the encoding gene, proC.

Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and delta1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0.93 nkat (mg protein)(-1) and 4.3 nkat (mg protein)(-1), respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28.9 kDa as calculated from the sequence. Apparent Km values for PCA and NADH were 0.19 mM and 0.025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent Km values: 1.55 mM for proline and 10.5 mM for NAD at pH 10.0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.

Amino Acid Sequence↗
PubMed1998

Fast purification of thioredoxin reductases and of thioredoxins with an unusual redox-active centre from anaerobic, amino-acid-utilizing bacteria.

Thioredoxin reductase and thioredoxin are primarily involved in catabolic metabolism as important electron carriers in anaerobic, amino-acid-degrading bacteria. A general and fast procedure was developed for the purification of thioredoxin reductase and thioredoxin from Eubacterium acidaminophilum, Clostridium litorale, C. sticklandii, C. sporogenes, C. cylindrosporum and 'Tissierella creatinophila' based upon their properties: the binding to 2',5'-AMP-Sepharose by thioredoxin reductase and the inability of thioredoxins to bind to a DEAE-Sephacel column. The consensus sequence at the active site of thioredoxins (-WCGPC-) was found to be modified in all of these anaerobes: Trp-31 (Escherichia coli nomenclature) was replaced by Gly or Ser, Gly-33 by Val or Glu. None of these thioredoxins reacted with thioredoxin reductase of E. coli or vice versa, but they did interact with the thioredoxin reductases obtained from the other anaerobes studied. Based upon their distinguishing features it is suggested that these thioredoxins might form an evolutionarily separate group.

Amino Acid Sequence↗