共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Yasuda K Jojima T Suda M Okino S Inui M Yukawa H 《Applied microbiology and biotechnology》2007,77(4):853-860
Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as
a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was
determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate
formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme
A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate
that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.
An erratum to this article can be found at 相似文献
3.
Wu QY Li F Zhu WJ Wang XY 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2007,148(4):355-362
Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. The gene encoding Locusta migratoria manilensis AK was cloned and expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 358 mg L− 1 of LB medium. Some parameters for the renatured and soluble forms of AK, including Km, Kd, specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest kcat and stronger synergistic substrate binding. The first report of a concise purification method enables the enzyme to be prepared in large quantities. This research should enable further detailed investigations of the enzymatic mechanism by site directed mutagenesis techniques. 相似文献
4.
The acetate kinase (ack) gene from Ethanoligenens sp. hit B49, isolated from a biohydrogen production bioreactor, is a key enzyme and responsible for dephosphorylation of acetyl phosphate
with the concomitant production of acetate and ATP; it was cloned, sequenced, and functionally expressed in Escherichia coli BL21(DE3). It contained a 1200-bp open reading frame and encoded a 399-amino-acid protein kinase (molecular weight, 43.22
kDa; isoionic point, pH 5.93) sharing 58% similarity with Thermotoga maritima MSB8 ack. Ack was heterologously expressed in E.coli BL21 (DE3). Ack specific activities of the refolded ack inclusion body from Ethanoligenens sp. hit B49 is 42.12 U at 25°C, and the renaturation percent is 14.36%. 相似文献
5.
Akio Tonouchi Yousuke Nishizaki Hiromi Tohyama Kiyoshi Takeda 《Current microbiology》2002,45(6):0390-0393
The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His6-tagged acetate kinase purified from E. coli JM109 was optimal at 35°C.
Received: 28 January 2002 / Accepted: 27 February 2002 相似文献
6.
G Ozcengiz J-H Kim W R Lin E Ozcengiz D Westenberg L R Lynd A L Demain 《Journal of industrial microbiology & biotechnology》1998,21(3):145-149
Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition
to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous
probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR-based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then successfully used as a probe for the isolation of the corresponding gene and restriction analysis of that
region.
Received 22 January 1998/ Accepted in revised form 31 August 1998 相似文献
7.
During growth ofCorynebacterium glutamicum on acetate as its carbon and energy source, the expression of theptaack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK).
By transposon rescue, we identified the two genesamrG1 andamrG2 found in the deregulated transposon mutant C.glutamicum G25. TheamrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially
responsible for the regulation of acetate metabolism in C.glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain ofamrG1 in the C.glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on
glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, theamrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities
of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion,
overexpression of theamrG1 gene in C.glutamicum 13032 had the adverse regulatory effect. These results indicate that theamrG1 gene encodes a repressor or co-repressor of theptaack operon. 相似文献
8.
During growth ofCorynebacterium glutamicum on acetate as its carbon and energy source, the expression of theptaack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK).
By transposon rescue, we identified the two genesamrG1 andamrG2 found in the deregulated transposon mutant C.glutamicum G25. TheamrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially
responsible for the regulation of acetate metabolism in C.glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain ofamrG1 in the C.glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on
glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, theamrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities
of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion,
overexpression of theamrG1 gene in C.glutamicum 13032 had the adverse regulatory effect. These results indicate that theamrG1 gene encodes a repressor or co-repressor of theptaack operon. 相似文献
9.
Michael Tyurin 《Journal of industrial microbiology & biotechnology》2013,40(7):749-758
A time- and cost-efficient two-step gene elimination procedure was used for acetogen Clostridium sp. MT1834 capable of fermenting CO2/H2 blend to 245 mM acetate (p < 0.005). The first step rendered the targeted gene replacement without affecting the total genome size. We replaced the acetate pta-ack cluster with synthetic bi-functional acetaldehyde-alcohol dehydrogenase (al-adh). Replacement of pta-ack with al-adh rendered initiation of 243 mM ethanol accumulation at the expense of acetate production during CO2/H2 blend continuous fermentation (p < 0.005). At the second step, al-adh was eliminated to reduce the genome size. Resulting recombinants accumulated 25 mM mevalonate in fermentation broth (p < 0.005). Cell duplication time for recombinants with reduced genome size decreased by 9.5 % compared to Clostridium sp. MT1834 strain under the same fermentation conditions suggesting better cell energy pool management in the absence of the ack-pta gene cluster in the engineered biocatalyst. If the first gene elimination step was used alone for spo0A gene replacement with two copies of synthetic formate dehydrogenase in recombinants with a shortened genome, mevalonate production was replaced with 76.5 mM formate production in a single step continuous CO2/H2 blend fermentation (p < 0.005) with cell duplication time almost nearing that of the wild strain. 相似文献
10.
Carole Colin Catherine Leblanc Gurvan Michel Elsa Wagner Emmanuelle Leize-Wagner Alain Van Dorsselaer Philippe Potin 《Journal of biological inorganic chemistry》2005,10(2):156-166
The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K m I– =2.5 mM; k cat I– =462 s–1) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K m I– =18.1 mM; k cat I– =38 s–1). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata
vIPO.The nucleotide sequence reported in this paper has been submitted to the EBI Data Bank with accession no. AJ619804. 相似文献
11.
Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate + ATP + CoA Acetyl-CoA + AMP + PPi). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41°C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37°C. Using the N-terminal amino acid sequence an open reading frame — coding for a 74 kDa protein — was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS. 相似文献
12.
Aspartate kinase (AK; EC 2.7.2.A) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids
in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations,Ask LT19 andAsk2 -LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to
characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived
amino acids in maize. Kernels of the homozygousAsk2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine,
compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 μMl-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygousAsk and homozygousAsk2 mutants required 25 and 760 μM lysine for 50% inhibition, respectively, indicating thatAsk andAsk2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the
homozygous mutantAsk2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutantAsk2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site
to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor ofAsk2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive
inhibition of wildtype AK. Reduced lysine sensitivity of theAsk2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygousAsk2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from
theAsk andAsk2 structural genes.
Scientific paper No. 17419, Minnesota Agricultural Experiment Station projects No. 0302-4813-56 and No. 0302-4818-32
This research was supported in part by the U.S. Depatment of Agriculture Competitive Research Grants Office grant 86-CRCR-1-2019.
The authors are grateful to Charles Grissom for providing the computer programs in an IBM-PC format. 相似文献
13.
Suzuki T Inoue N Higashi T Mizobuchi R Sugimura N Yokouchi K Furukohri T 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2000,127(4):505-512
Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a Km value of 0.6 mM for arginine and a Vmax value of 13 μmole Pi min−1 mg protein−1 for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1608 and 1239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66–73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63–71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55–57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered. 相似文献
14.
Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K
i=0.5 mM), by azide (apparent K
i=1 mM), and by cyanide (apparent K
i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H2/CO2 grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO2 and CH4. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO2 with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein). 相似文献
15.
Dawson NJ Storey KB 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2011,181(7):851-859
Freshwater crayfish, Orconectes virilis, can experience periodic exposures to hypoxia or anoxia due to low water flow (in summer) or ice cover (in winter) in their
natural habitat. Hypoxia/anoxia disrupts energy metabolism and triggers mechanisms that to support ATP levels while often
also suppressing ATP use. Arginine kinase (AK) (E.C. 2.7.3.3) is a crucial enzyme involved in energy metabolism in muscle,
gating the use of phosphagen stores to buffer ATP levels. The present study investigated AK from tail muscle of O. virilis identifying changes to kinetic properties, phosphorylation state and structural stability between the enzyme from aerobic
control and 20 h anoxic crayfish. Muscle AK from anoxia-exposed crayfish showed a significantly higher (by 59%) K
m for l-arginine and a lower I50 value for urea than the aerobic form. Several lines of evidence indicated that AK was converted to a high phosphate form
under anoxia: (a) aerobic and anoxic forms of AK showed well-separated elution peaks on DEAE ion exchange chromatography,
(b) ProQ Diamond phosphoprotein staining showed a 64% higher bound phosphate content on anoxic AK compared with the aerobic
form, and (c) treatment of anoxic AK with alkaline phosphatase reduced K
m
l-arginine to aerobic levels whereas incubation of aerobic AK with protein kinase A catalytic subunit raised the K
m to anoxic levels. The physiological consequence of anoxia-induced AK phosphorylation may be to suppress AK activity in the
phosphagen-synthesizing direction and, together with reduced cellular pH and ATP levels, promote the phosphagen-catabolizing
direction under anoxic conditions. This is first time that AK has been shown to be regulated by reversible phosphorylation. 相似文献
16.
Esteban Sánchez Pablo C. Garcia Luis R. López-Lefebre Rosa M. Rivero Juan Manuel Ruiz Luis Romero 《Plant Growth Regulation》2002,36(3):261-265
The objective of the present work was to determine the impact ofnitrogen deficiency on proline metabolism in French Bean plants(Phaseolus vulgaris L. cv. Strike). The nitrogen wasapplied to the nutrient solution in the form of NH4NO3 at1.45 mM (N1), 2.90mM (N2) and 5.80mM (N3, optimal level). Our results indicateNdeficiency is characterised by a decline in proline accumulation both in theroot and leaves, fundamentally because proline degradation is encouraged by thestimulation of the enzyme proline dehydrogenase. By contrast, under conditionsof adequate N (N3), proline levels rise due to the action of ornithine,suggesting predominance of the ornithine pathway over the glutamine pathway, inaddition to the inhibition of proline dehydrogenase activity. 相似文献
17.
Tatsuya Ohshida Kohei Koba Junji Hayashi Kazunari Yoneda Taketo Ohmori Toshihisa Ohshima 《Bioscience, biotechnology, and biochemistry》2013,77(12):2084-2093
ABSTRACTThe orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine.Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK–HseDH: bifunctional aspartate kinase–homoserine dehydrogenase; AsaDH: aspartate–β–semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T). 相似文献
18.
V. N. R. Kartha N. Jayanthi Bai Thomas George S. Krishnamurthy 《Journal of biosciences》1979,1(2):159-168
An enzyme catalysing the hydrolysis ofa-tocopheryl acetate was characterised in chicken liver. The enzyme was localised in the microsomes, had an optimum pH 8.6
and aK
m value of 0.5 mM. The enzyme did not hydrolyse retinyl acetate, cholesteryl acetate and ethyl acetate, thus indicating a high
degree of specificity.a-Tocopheryl acetate hydrolase required bile salts as a specific co-factor. The results suggested a role for this enzyme in
the absorption of vitamin E. 相似文献
19.
Identification,characterization, and comparison of RNA-degrading enzymes of wheat and barley 总被引:1,自引:0,他引:1
RNA-degrading enzymes play an important role in regulating gene expression, and sequence analyses have revealed significant
homology among several plant RNA-degrading enzymes. In this study we surveyed crude extracts of the above-ground part of the
common wheat (Triticum aestivum L.) and the cultivated barley (Hordeum vulgare L.) for major RNA-degrading enzymes using a substrate-based SDS-PAGE assay. Fifteen wheat and fourteen barley RNA-degrading
enzymes, with apparent molecular masses ranging from 16.3 to 40.1 kD, were identified. These RNA-degrading enzymes were characterized
by their response to pH changes and addition of EDTA and ZnCl2 to the preincubation or incubation buffers. The 33.2- to 40.1-kD wheat and barley, 31.7-kD wheat, and 32.0-kD barley enzyme
activities were inhibited by both zinc and EDTA and were relatively tolerant to alkaline environment. The 22.7- to 28.2-kD
enzymes were inhibited by zinc but stimulated by EDTA. The 18.8-kD enzyme exists in both wheat and barley. It was active in
an acid environment, was inhibited by zinc, but was not affected by EDTA. Two enzyme activities (31.0 and 32.0 kD) are unique
to the common wheat.
Contribution from Agriculture Research Division, University of Nebraska, Journal Series No. 9895. 相似文献
20.
A ureidoglycolate-degrading activity was analysed in different tissues of French bean (Phaseolus vulgaris L.) plants during development. Activity was detected in all the tissues analysed, although values were very low in seeds before germination and in cotyledons. After radicle emergence, the activity increased due to high activity present in the axes. The highest levels of specific activity were found in developing fruits, from which the enzyme was purified and characterised. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from a ureide legume. The enzyme was purified 280 fold, and the specific activity for the pure enzyme was 4.4 units mg−1, which corresponds to a turnover number of 1,055 min−1. The native enzyme has a molecular mass of 240 kDa and consists of six identical or similar-sized subunits each of 38 kDa. The activity of the purified enzyme was completely dependent on manganese and asparagine. The enzyme exhibited hyperbolic, Michaelian kinetics for ureidoglycolate with a K
m value of 3.9 mM. This enzyme has been characterised as a ureidoglycolate urea-lyase (EC 4.3.2.3). 相似文献