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ABSTRACT: Prokaryotic ubiquitin-like protein (Pup) is a functional analog of ubiquitin. Post-translationally modified pupylated proteins are selectively degraded by a proteasome-dependent proteolytic system. Deaminase of Pup (Dop) activates Pup by deaminating the C-terminal from glutamine to glutamate, and subsequently activated Pup is conjugated to target proteins by proteasome accessory factor A. Dop is also involved in the removal of Pup from pupylated proteins. Deconjugated free Pup is capable of religating to target proteins. Although the pupylation system is well studied in Mycobacterium, little is known about it in other actinomycetes. Both Rhodococcus and Mycobacterium Dop remove Pup from pupylated proteins, but in these two bacteria, no accumulation of deconjugated free Pup from Rhodococcus is observed. Analysis of a model pupylated protein revealed that Rhodococcus Pup is degraded at multiple sites by Dop. The endopeptidase activity of Dop can be detected using a fluorogenic substrate in conjunction with aminopeptidase. Moreover, the enzymatic activity of the model enzyme increases when Pup is deconjugated. These results suggest that depupylated Rhodococcus Pup is not recycled for religation with target proteins, and that Pup not only functions as a degradation signal, but also regulates the enzymatic activity of target proteins by conjugation and deconjugation to them.
Bioscience Biotechnology and Biochemistry 10/2012; 76(10):1959-66. · 1.28 Impact Factor
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ABSTRACT: The genus Rhodococcus exhibits a broad range of catalytic activity and is tolerant to various kinds of organic solvents. This property makes rhodococci suitable for use as a whole-cell catalyst. Various tools for genetic engineering have been developed to use Rhodococcus erythropolis as a host for bioconversion. In this study, we investigated the protein expression responses of R. erythropolis strains and found that isocitrate lyase production in R. erythropolis PR4 (ICL(Re)) was induced by methanol. By analyzing the regulation mechanisms of icl(Re) expression, the ~200-bp upstream region from the first nucleotide of the translation initiation codon of icl(Re) was shown to be sufficient for the methanol-inducible expression. Also, the ~100-bp upstream region exhibited strong constitutive promoter activity by an unknown mechanism(s). By investigating proteins that bound to the upstream region of icl(Re)in vitro, a RamB homologue of R. erythropolis PR4 (RamB(Re)) was identified. Moreover, 2 putative RamB(Re) binding sites were identified in the upstream region of icl(Re) through pull-down assays. A ramB(Re) knockout experiment suggested that RamB(Re) negatively controlled the expression of icl(Re) and that RamB(Re) regulation was dependent on the availability of a carbon source. On the basis of these findings, we were able to create novel methanol-inducible and strong constitutive expression vectors.
Journal of Bioscience and Bioengineering 01/2012; 113(5):596-603. · 1.79 Impact Factor
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ABSTRACT: We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D(3) hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.
Applied and environmental microbiology 02/2010; 76(8):2531-9. · 3.69 Impact Factor
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Masao Doisaki,
Yoshiaki Katano,
Isao Nakano,
Yoshiki Hirooka,
Akihiro Itoh,
Masatoshi Ishigami,
Kazuhiko Hayashi,
Hidemi Goto,
Yuko Fujita,
Yoshihiro Kadota,
Yasuyuki Kitaura,
Gustavo Bajotto,
Shunsuke Kazama,
Tomohiro Tamura, Noriko Tamura,
Guo-Gang Feng,
Naohisa Ishikawa,
Yoshiharu Shimomura
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ABSTRACT: Branched-chain alpha-keto acid dehydrogenase (BCKDH) kinase (BDK) is responsible for the regulation of BCKDH complex, which is the rate-limiting enzyme in the catabolism of branched-chain amino acids (BCAAs). In the present study, we investigated the expression and activity of hepatic BDK in spontaneous type 2 diabetes using hyperinsulinemic Zucker diabetic fatty rats aged 9weeks and hyperglycemic, but not hyperinsulinemic rats aged 18weeks. The abundance of hepatic BDK mRNA and total BDK protein did not correlate with changes in serum insulin concentrations. On the other hand, the amount of BDK bound to the complex and its kinase activity were correlated with alterations in serum insulin levels, suggesting that hyperinsulinemia upregulates hepatic BDK. The activity of BDK inversely corresponded with the BCKDH complex activity, which was suppressed in hyperinsulinemic rats. These results suggest that insulin regulates BCAA catabolism in type 2 diabetic rats by modulating the hepatic BDK activity.
Biochemical and Biophysical Research Communications 02/2010; 393(2):303-7. · 2.48 Impact Factor
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Seikagaku. The Journal of Japanese Biochemical Society 10/2009; 81(10):896-9. · 0.04 Impact Factor
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ABSTRACT: We used molecular sieve chromatography in combination with LC-MS/MS to identify protein complexes that can serve as templates in the template matching procedures of visual proteomics approaches. By this method the sample complexity was lowered sufficiently to identify 464 proteins and - on the basis of size distribution and bioinformatics analysis - 189 of them could be assigned as subunits of macromolecular complexes over the size of 300 kDa. From these we purified six stable complexes of Thermoplasma acidophilum whose size and subunit composition - analyzed by electron microscopy and MALDI-TOF-MS, respectively - verified the accuracy of our method.
Proteomics 08/2009; 9(14):3783-6. · 4.43 Impact Factor
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ABSTRACT: Vitamin D(3) (VD(3)) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD(3) into its physiologically active forms, namely, 25(OH)VD(3) or 1alpha,25(OH)(2)VD(3). In this study, we isolated and characterized Vdh (vitamin D(3) hydroxylase), which hydroxylates VD(3) from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V(max) values for VD(3) 25-hydroxylation and 25(OH)VD(3) 1alpha-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD(3). We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD(3) 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD(3) into 25(OH)VD(3) was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD(3) by a single cytochrome P450.
Biochemical and Biophysical Research Communications 06/2009; 385(2):170-5. · 2.48 Impact Factor
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ABSTRACT: The D-aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D-aldohexoses, especially D-mannose. AldT comprises a unique C-terminal tail motif (residues 247-255) that shuts the active-site pocket of the neighboring subunit. The functional role of the C-terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C-terminal deletion mutants (Delta254, Delta253, Delta252, and Delta249) and two site-specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C-terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Delta249 mutant was also determined. The structure showed that the active-site loops undergo a significant conformational change, which leads to the structural deformation of the substrate-binding pocket.
Proteins Structure Function and Bioinformatics 11/2008; 74(4):801-7. · 3.39 Impact Factor
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Teiji Kuzuya,
Yoshiaki Katano,
Isao Nakano,
Yoshiki Hirooka,
Akihiro Itoh,
Masatoshi Ishigami,
Kazuhiko Hayashi,
Takashi Honda,
Hidemi Goto,
Yuko Fujita,
Rie Shikano,
Yuji Muramatsu,
Gustavo Bajotto,
Tomohiro Tamura, Noriko Tamura,
Yoshiharu Shimomura
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ABSTRACT: The branched-chain alpha-keto acid dehydrogenase (BCKDH) complex is the most important regulatory enzyme in branched-chain amino acid (BCAA) catabolism. We examined the regulation of hepatic BCKDH complex activity in spontaneous type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Zucker diabetic fatty rats. Hepatic BCKDH complex activity in these rats was significantly lower than in corresponding control rats. The amount of BCKDH complex in OLETF rats corresponded to the total activity of the complex. Activity and abundance of the bound form of BCKDH kinase, which is responsible for inactivation of the complex, showed an inverse correlation to BCKDH complex activity in OLETF rats. Dietary supplementation of 5% BCAAs for 10 weeks markedly increased BCKDH complex activity, and decreased the activity and bound form of BCKDH kinase in the rats. These results suggest that BCAA catabolism in type 2 diabetes is downregulated and enhanced by BCAA supplementation.
Biochemical and Biophysical Research Communications 09/2008; 373(1):94-8. · 2.48 Impact Factor
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ABSTRACT: Adipocytes are now recognized as endocrine cells secreting adipocytokines, regulating multiple metabolic pathways. In this study, we addressed secretion of microvesicles by 3T3-L1 adipocytes. We found that MFG-E8, one of the exosomal proteins, was present in the microvesicles and was distributed in the sucrose density fractions with 1.13-1.20 g/ml, which has been reported for exosomes. Several integral, cytosolic, and nuclear proteins such as caveolin-1, c-Src kinase, and heat shock protein 70 were also found to be microvesicle components. Unexpectedly, adiponectin was also substantially distributed in the microvesicle fractions. Furthermore, proteomic analysis of the microvesicles revealed that many other proteins such as extracellular matrix-related proteins were also present. Microvesicles secreted by 3T3-L1 adipocytes exhibited heterogeneity in size and comprised both smaller exosome-like and larger membrane vesicles as revealed by electron microscopy. Milk fat globule-epidermal growth factor 8 (MFG-E8)-associated adiposomes exhibited binding activity toward phosphatidylserine and apoptotic cells. MFG-E8 in the microvesicles was reduced when cultured in the low-glucose medium or cultured in the high-glucose medium with antioxidant N-acetyl cysteine. Insulin and TNF-alpha also up-regulated MFG-E8 in the microvesicles. Moreover, MFG-E8 was strongly up-regulated in the hypertrophic adipose tissue, predominantly in adipocyte fractions, of diet-induced obese C57BL/6 mice, where increased oxidative stress is induced. Thus, it is suggested that microvesicles, especially MFG-E8-associated ones, modulate adipose functions under redox- and hormone-dependent regulation. Based on the above findings, the adipocyte-derived microvesicles were named adiposomes.
Endocrinology 09/2007; 148(8):3850-62. · 4.46 Impact Factor
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ABSTRACT: The D-aldohexose dehydrogenase from the thermoacidophilic archaea Thermoplasma acidophilum (AldT) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and catalyzes the oxidation of several monosaccharides with a preference for NAD(+) rather than NADP(+) as a cofactor. It has been found that AldT is a unique enzyme that exhibits the highest dehydrogenase activity against D-mannose. Here, we describe the crystal structures of AldT in ligand-free form, in complex with NADH, and in complex with the substrate D-mannose, at 2.1 A, 1.65 A, and 1.6 A resolution, respectively. The AldT subunit forms a typical SDR fold with an unexpectedly long C-terminal tail and assembles into an intertwined tetramer. The D-mannose complex structure reveals that Glu84 interacts with the axial C2 hydroxyl group of the bound D-mannose. Structural comparison with Bacillus megaterium glucose dehydrogenase (BmGlcDH) suggests that the conformation of the glutamate side-chain is crucial for discrimination between D-mannose and its C2 epimer D-glucose, and the conformation of the glutamate side-chain depends on the spatial arrangement of nearby hydrophobic residues that do not directly interact with the substrate. Elucidation of the D-mannose recognition mechanism of AldT further provides structural insights into the unique substrate selectivity of AldT. Finally, we show that the extended C-terminal tail completely shuts the substrate-binding pocket of the neighboring subunit both in the presence and absence of substrate. The elaborate inter-subunit interactions between the C-terminal tail and the entrance of the substrate-binding pocket imply that the tail may play a pivotal role in the enzyme activity.
Journal of Molecular Biology 05/2007; 367(4):1034-46. · 4.00 Impact Factor
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ABSTRACT: In the proteolytic pathway of prokaryotic and eukaryotic organisms, proteins tagged for proteolysis are firstly shredded into smaller peptides by compartmentalized proteases such as the proteasome complex. Accordingly, a variety of downstream proteases have evolved to further hydrolyze these peptides to the level of free amino acids. In the search for such downstream proteases, a high-molecular-weight protease complex called trilobed protease (TLP) was recently discovered in the archaeon Pyroccocus furiosus. The crystal structure of the N-terminal beta-propeller domain of the trilobed protease at 2 A resolution shows that the trilobed protease utilizes this accessory domain to control substrate access to the active site. Modelling of the intact TLP monomer suggests that this protease has an additional side entrance to its active site as in the DPP-IV or tricorn protease complexes.
Acta Crystallographica Section D Biological Crystallography 03/2007; 63(Pt 2):179-87. · 12.62 Impact Factor
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ABSTRACT: In the current study we developed two transposon-based vectors; namely pTNR-KA and pTNR-TA and utilized them for expression of proteasome complex, derived from Streptomyces coelicolor, in Rhodococcus erythropolis. The two vectors can be transposed into Rhodococcus cells by means of electroporation, either individually in two consecutive processes or in combinations by a single step. During transposition, each of the two vectors liberates its transposable-marker gene, which integrated in a single copy into a random site in the Rhodococcus chromosomal DNA. Southern blot analysis indicated that the two transposable-marker genes of both vectors does not alter or knock out each other. To utilize these vectors for Streptomyces proteasome expression, two expression cassettes were constructed; each cassette comprised a constitutive promoter (P(nit)), the DNA fragment, prcA or prcB that encodes alpha- or beta-subunits of Streptomyces proteasome, and T(thcA) transcriptional terminator. The cassettes were then individually introduced into the multiple cloning sites that are located in the transposable-marker gene of the two vectors. The two cassettes-harboring vectors were subsequently co-transposed, in combinations, into the Rhodococcus genome by a single electroporation step and the Streptomyces proteasome was successfully expressed in the rodococcal host cell. The isolated proteasome was further characterized and the peptidase activity was confirmed and indicated that it was biologically active. The present study concluded that both pTNR-KA and pTNR-TA can be used as transposon-based protein expression systems in Rhodococcus species.
Gene 02/2007; 386(1-2):173-82. · 2.34 Impact Factor
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ABSTRACT: The aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a 28 kDa molecular-weight enzyme that catalyzes the oxidation of various aldohexoses, with a preference for NAD+ rather than NADP+ as a cofactor. The recombinant AldT was crystallized using the hanging-drop vapour-diffusion technique at 293 K under several acidic conditions with polyethylene glycol (PEG) and ammonium sulfate as precipitants. Optimization of the initial crystallizations conditions yielded single crystals in solution containing 0.1 M sodium acetate pH 4.6, 18%(w/v) PEG 4000, 0.2 M ammonium sulfate and 15%(v/v) glycerol. An X-ray diffraction data set was collected to a resolution of 2.8 A.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2006; 62(Pt 6):586-9. · 0.51 Impact Factor
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ABSTRACT: Branched-chain amino acid leucine has been shown to activate the translational regulators through the mammalian target of rapamycin. However, the leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway. The irreversible and rate-limiting step in the leucine oxidation pathway is catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKDH) complex. The complex contains E1 (alpha2beta2), E2, and E3 subunits, and its activity is abolished by phosphorylation of the E1alpha subunit by BCKDH kinase. The relationship between the activity of BCKDH complex and leucine-mediated activation of the protein translation was investigated using the technique of RNA interference. The activity of BCKDH complex in C2C12 cell was modulated by transfection of small interfering RNA (siRNA) for BCKDH E2 subunit or BCKDH kinase. Transfection of siRNAs decreased the mRNA expression and protein amount of corresponding gene. Suppression of either E2 subunit or kinase produced opposite effects on the cell proliferation and the activation of translational regulators by leucine. Suppression of BCKDH kinase for 48h resulted in decreasing cell proliferation. In contrast, E2 suppression led to increased amount of total cellular protein. The phosphorylation of p70 S6 kinase by leucine was increased in E2-siRNA transfected C2C12 cells, whereas the leucine's effect was diminished in kinase-siRNA transfected cells. These results suggest that the activation of the translational regulators by leucine was partly regulated by the activity of BCKDH complex.
Biochemical and Biophysical Research Communications 06/2006; 343(4):1244-50. · 2.48 Impact Factor
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ABSTRACT: Tumor necrosis factor-alpha (TNFalpha) promotes oxidation of branched-chain amino acids (BCAA). BCAA catabolism is regulated by branched-chain alpha-keto acid dehydrogenase (BCKDH) complex, which is regulated by phosphorylation-dephosphorylation of the E1alpha subunit at Ser293. BCKDH kinase is responsible for inactivation of the complex by phosphorylation. In the present study, we examined the effects of TNFalpha administration on hepatic BCKDH complex and kinase in rats. Rats were intravenously administered with 25 or 50 microg TNFalpha/kg body weight 4 h prior to sacrifice. The TNFalpha treatment at both doses elevated the activity state (percentage of the active form) of BCKDH complex from 22% to 69% and 86%, respectively, and the amount of phospho-Ser293 on the E1alpha subunit in each group of rats corresponded inversely to the activity state of BCKDH complex. The TNFalpha treatment of rats significantly decreased the activity as well as the bound form of BCKDH kinase. These results suggest that the decrease in the bound form of kinase is involved in the mechanism responsible for TNFalpha-induced activation of the BCKDH complex.
Biochemical and Biophysical Research Communications 04/2005; 328(4):973-8. · 2.48 Impact Factor
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ABSTRACT: The NADP(+)-preferring glucose dehydrogenase from thermoacidophilic archaeon Thermoplasma acidophilum has been characterized, and its crystal structure has been determined (Structure, 2:385-393, 1994). Its sequence and structure are not homologous to bacterial NAD(P)(+)-dependent glucose dehydrogenases, and its molecular weight is also quite defferent. On the other hand, three functionally unknown genes with homologies to bacterial NAD(P)(+)-dependent glucose dehydrogenases have been sequenced as part of the T. acidophilum genome project (gene names: Ta0191, Ta0747, and Ta0754 respectively). We expressed two genes of three, Ta0191 and Ta0754, in Escherichia coli, and purified the gene products to homogeneity. Dehydrogenase activities were thereby detected from the purified proteins. The Ta0754 gene product exhibited aldohexose dehydrogenase activity, and the Ta0191 gene product exhibited weak 2-deoxyglucose dehydrogenase activity. No aldohexose dehydrogenase gene has been isolated, while the enzyme was reported in 1968. This is the first report of the gene and primary structure. The purified Ta0754 gene product, designated AldT, was characterized. The enzyme AldT effectively catalyzed the oxidation of various aldohexoses, especially D-mannose. Lower activities on D-2-deoxyglucose, D-xylose, D-glucose, and D-fucose were detected although no activities were shown on other aldohexoses or additional sugars. As a cofactor, NAD(+) was much more suitable for the activity than NADP(+). The NAD(+)-preferring dehydrogenase most effectively reacting to D-mannose is for the first time. AldT was most active at pH 10 and above 70 degrees C, and completely stable up to 60 degrees C after incubation for 15 min. Other enzymatic properties were also investigated.
Bioscience Biotechnology and Biochemistry 01/2005; 68(12):2451-6. · 1.28 Impact Factor
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ABSTRACT: The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidative decarboxylation of pyruvate in mitochondria. The PDC activity is regulated by a phosphorylation/dephosphorylation cycle catalyzed by specific kinases (PDK) and phosphatases (PDP). In this study, the regulatory mechanisms of PDC were examined in skeletal muscle of the spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rat before and after the onset of diabetes. The Long-Evans Tokushima Otsuka (LETO) rat was used as control. Plasma glucose and insulin concentrations were at normal levels in both groups at 8 weeks of age but were significantly higher in OLETF than in LETO rats at 25 weeks of age (1.2-fold for glucose and 15-fold for insulin), indicating development of diabetes in the former. Plasma free fatty acids were 1.6-fold concentrated and the skeletal muscle PDC activity state was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidation in OLETF rats even before the onset of diabetes. The PDK activity and the abundance of the PDK isoform 4 protein as well as mRNA were greater in OLETF rats at both ages. Conversely, the abundance of the PDP isoform 1 protein and mRNA was less in OLETF than in LETO rats at both ages. These results suggest that concomitant greater PDK4 and less PDP1 expression in skeletal muscle of OLETF rats before the onset of diabetes are responsible for the lowering of the PDC activity and may be related with the development of diabetes mellitus.
Life Sciences 10/2004; 75(17):2117-30. · 2.53 Impact Factor
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ABSTRACT: The Lon protease from the archaeon Thermoplasma acidophilum (TaLon) is composed of an N-terminal ATPase associated with various cellular activities (AAA+) domain and a C-terminal Lon protease domain. Although related in sequence to the soluble Lon proteases, TaLon was shown to be membrane-bound in its native host and also when expressed in Escherichia coli. Recombinant TaLon was purified as a functional high-molecular weight complex displaying ATPase and proteolytic activity. Mutagenesis of conserved AAA+ residues revealed that the Walker A and B motifs, and the sensor 1 and sensor 2' residues were essential for the ATPase activity, while the sensor 2 and the arginine finger were involved in activation of the protease domain.
FEBS Letters 10/2004; 574(1-3):161-6. · 3.54 Impact Factor
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ABSTRACT: We cloned the Rhodococcus opacus (strain DSM 44193) tipA gene, which encodes two translation products, TipAL and TipAS. The gene products are homologous to the Streptomyces spp. TipAL and TipAS proteins, respectively. The tipA promoter is highly active and TipAS protein is predominantly accumulated in R. opacus cells when the inducer of transcription, thiostrepton, was presented in culture medium. We found that thiostrepton is also induced the expression of an endogenous TipA-family protein in Rhodococcus erythropolis (strain JCM3201). The minimal tipA promoter region was defined (57 bp) and the conserved nucleotide sequence of the putative TipAL protein binding site (TipA-box) was identified in that region. The tipA gene is presumed to be transcribed into a leaderless mRNA. We applied the tipA promoter successfully for recombinant protein expression in R. erythropolis cells.
FEMS Microbiology Letters 09/2004; 237(1):35-40. · 2.04 Impact Factor
