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1.
Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PPi. Crystal structures of metal-free Duf89 and Co2+-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl2 and NaF in the presence of divalent cations Co2+, Ni2+, or Zn2+ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF3)•Co2+, Duf8901-(Asp248)–BeF3•Ni2+, or Duf8901-(Asp248)–BeF3•Zn2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF3•Co2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF4–OH2•Co2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo. 相似文献
2.
Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes 总被引:1,自引:0,他引:1
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Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp-PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pHv) of 4.74 ± 0.1 is generated in the presence of 0.1 millimolar PPi, but only 4.93 ± 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PPi. Cl−-stimulates the H+-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K½ ≈ 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PPi. The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pHv unit has been estimated. From the free energy (−11.42 kilojoules per mole) for the hydrolysis of PPi under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H+ per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H+-pump rather than as a pyrophosphate synthetase. 相似文献
3.
Youské Shimizu Hitoshi Sakakibara Hiroaki Kojima Kazuhiro Oiwa 《Biophysical journal》2014,106(10):2157-2165
We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s–1 and kcat,MT = 1.09 s–1, respectively) were lower than those of dynein c (kcat = 1.75 s–1 and kcat,MT = 2.03 s–1, respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (Vap = 1.2 ± 0.1 μm/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (Vmax = 15.8 ± 1.5 μm/s) and highly sensitive to decreases in the surface density (Vmin = 2.2 ± 0.7 μm/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c’s power stroke. 相似文献
4.
Jean-Marie Frère Jean-Marie Ghuysen Peter E. Reynolds Ramon Moreno Harold R. Perkins 《The Biochemical journal》1974,143(1):241-249
Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl2 buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10−6, 1.5×10−6 and 0.63×10−6s−1 (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [14C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [14C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10−5s−1 and 0.8×10−4s−1 respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site. 相似文献
5.
Reinaldo DiPolo 《The Journal of general physiology》1974,64(4):503-517
Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm-2·s-1 (P/CS) (n = 8) at 22°C. With [ATP]i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]i. An increase of the calcium efflux is observed when [ATP]i is >100 µM and saturates at [ATP]i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na+, Ca++, and Mg++. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations. 相似文献
6.
Jerneja Kladnik Ana Dolinar Jakob Kljun David Perea Judith Grau-Expsito Meritxell Genesc Marko Novinec Maria J. Buzon Iztok Turel 《Journal of enzyme inhibition and medicinal chemistry》2022,37(1):2158
Zinc pyrithione (1a), together with its analogues 1b–h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC50=1.88 ± 0.49 µM) and PLPro (IC50=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b–h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2. 相似文献
7.
Lauren J. Sundby William M. Southern Katelin M. Hawbaker Jesús M. Trujillo Benjamin J. Perrin James M. Ervasti 《Molecular biology of the cell》2022,33(9)
Cytoplasmic β- and γ-actin proteins are 99% identical but support unique organismal functions. The cytoplasmic actin nucleotide sequences Actb and Actg1, respectively, are more divergent but still 89% similar. Actb–/– mice are embryonic lethal and Actb–/– cells fail to proliferate, but editing the Actb gene to express γ-actin (Actbc–g) resulted in none of the overt phenotypes of the knockout revealing protein-independent functions for Actb. To determine if Actg1 has a protein-independent function, we crossed Actbc–g and Actg1–/– mice to generate the bG/0 line, where the only cytoplasmic actin expressed is γ-actin from Actbc–g. The bG/0 mice were viable but showed a survival defect despite expressing γ-actin protein at levels no different from bG/gG with normal survival. A unique myopathy phenotype was also observed in bG/0 mice. We conclude that impaired survival and myopathy in bG/0 mice are due to loss of Actg1 nucleotide-dependent function(s). On the other hand, the bG/0 genotype rescued functions impaired by Actg1–/–, including cell proliferation and auditory function, suggesting a role for γ-actin protein in both fibroblasts and hearing. Together, these results identify nucleotide-dependent functions for Actg1 while implicating γ-actin protein in more cell-/tissue-specific functions. 相似文献
8.
We obtained detailed kinetic characteristics–stoichiometry, reaction rates, substrate affinities and equilibrium conditions–of human PPIP5K2 (diphosphoinositol pentakisphosphate kinase 2). This enzyme synthesizes ‘high-energy’ PP-InsPs (diphosphoinositol polyphosphates) by metabolizing InsP6 (inositol hexakisphosphate) and 5-InsP7 (5-diphosphoinositol 1,2,3,4,6-pentakisphosphate) to 1-InsP7 (1-diphosphoinositol 2,3,4,5,6-pentakisphosphate) and InsP8 (1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate), respectively. These data increase our insight into the PPIP5K2 reaction mechanism and clarify the interface between PPIP5K catalytic activities and cellular bioenergetic status. For example, stochiometric analysis uncovered non-productive, substrate-stimulated ATPase activity (thus, approximately 2 and 1.2 ATP molecules are utilized to synthesize each molecule of 1-InsP7 and InsP8, respectively). Impaired ATPase activity of a PPIP5K2-K248A mutant increased atomic-level insight into the enzyme''s reaction mechanism. We found PPIP5K2 to be fully reversible as an ATP-synthase in vitro, but our new data contradict previous perceptions that significant ‘reversibility’ occurs in vivo. PPIP5K2 was insensitive to physiological changes in either [AMP] or [ATP]/[ADP] ratios. Those data, together with adenine nucleotide kinetics (ATP Km=20–40 μM), reveal how insulated PPIP5K2 is from cellular bioenergetic challenges. Finally, the specificity constants for PPIP5K2 revise upwards by one-to-two orders of magnitude the inherent catalytic activities of this enzyme, and we show its equilibrium point favours 80–90% depletion of InsP6/5-InsP7. 相似文献
9.
Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA
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In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔHvHpm = 18.0 ± 1.6 kcal·mol–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔHvHpm = 13.4 ± 2.5 kcal·mol–1) differ by an amount (~4.6 kcal·mol–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)n·(dT)n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·molbp–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·molbp–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔGst = 0.8 ± 0.3 kcal· molbp–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔGst = 0.3 ± 0.3 kcal·molbp–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔHvHA = 407 ± 23 kcal·mol–1, ΔSvHA = 1166 ± 67 cal·°K–1·mol–1, ΔGvH(25°C)A = 60.0 ± 3.2 kcal·mol–1) are clearly distinguished from those of T (ΔHvHT = 185 ± 38 kcal·mol–1, ΔSvHT = 516 ± 109 cal·°K–1·mol–1, ΔGvH(25°C)T = 27.1 ± 5.5 kcal·mol–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔHvHA = 333 ± 54 kcal·mol–1, ΔSvHA = 961 ± 157 cal·°K–1·mol–1, ΔGvH(25°C)A = 45.0 ± 7.6 kcal· mol–1; ΔHvHT = 213 ± 30 kcal·mol–1, ΔSvHT = 617 ± 86 cal·°K–1·mol–1, ΔGvH(25°C)T = 29.3 ± 4.9 kcal·mol–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation. 相似文献
10.
Suzuki T Ide H Yamada M Endo N Kanaori K Tajima K Morii T Makino K 《Nucleic acids research》2000,28(2):544-551
The reaction mechanism for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by nitrous acid was explored using methyl derivatives of guanosine and an isolated intermediate of the reaction. When 1-methylguanosine was incubated with NaNO2 under acidic conditions, N5-methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N2,N2-dimethylguanosine generated no product. In a similar experiment without NO2–, participation of a Dimroth rearrangement was ruled out. In the guanosine–HNO2 reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20°C) was isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was converted into oxanosine and xanthosine at a molar ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and 20°C. The ratio was not affected by the incubation pH between 2 and 10, but increased linearly with temperature from 0.22 (0°C) to 0.32 (50°C). The addition of acetone also increased the ratio up to 0.85 (98% acetone). Based on these results, a con-ceivable pathway for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by HNO2 is proposed. 相似文献
11.
12.
Kazuma Okada Ryota Hidese Wakao Fukuda Masaru Niitsu Koichi Takao Yuhei Horai Naoki Umezawa Tsunehiko Higuchi Tairo Oshima Yuko Yoshikawa Tadayuki Imanaka Shinsuke Fujiwara 《Journal of bacteriology》2014,196(10):1866-1876
Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N4-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N4-bis(aminopropyl)spermidine, an isomer of N4-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N4-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N4-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap–time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N4-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N4-bis(aminopropyl)spermidine from spermidine via N4-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N4-aminopropylspermidine and N4-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments. 相似文献
13.
Yun-Tzu Huang Tseng-Huang Liu Yen-Wei Chen Chien-Hsien Lee Hsueh-Hua Chen Tsu-Wei Huang Shen-Hsing Hsu Shih-Ming Lin Yih-Jiuan Pan Ching-Hung Lee Ian C. Hsu Fan-Gang Tseng Chien-Chung Fu Rong-Long Pan 《The Journal of biological chemistry》2010,285(31):23655-23664
Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding. 相似文献
14.
Yaohui Shi Guangsheng Zhou Yanling Jiang Hui Wang Zhenzhu Xu Jian Song 《Ecology and evolution》2015,5(14):2956-2965
Nitrogen (N) serves as an important mineral element affecting plant productivity and nutritional quality. However, few studies have addressed the interactive effects of elevated CO2 and precipitation change on leaf N of dominant grassland genera such as Stipa L. This has restricted our understanding of the responses of grassland to climate change. We simulated the interactive effects of elevated CO2 concentration and varied precipitation on leaf N concentration (Nmass) of four Stipa species (Stipa baicalensis, Stipa bungeana, Stipa grandis, and Stipa breviflora; the most dominant species in arid and semiarid grassland) using open-top chambers (OTCs). The relationship between the Nmass of these four Stipa species and precipitation well fits a logarithmic function. The sensitivity of these four species to precipitation change was ranked as follows: S. bungeana > S. breviflora > S. baicalensis > S. grandis. The Nmass of S. bungeana was the most sensitive to precipitation change, while S. grandis was the least sensitive among these Stipa species. Elevated CO2 exacerbated the effect of precipitation on Nmass. Nmass decreased under elevated CO2 due to growth dilution and a direct negative effect on N assimilation. Elevated CO2 reduced Nmass only in a certain precipitation range for S. baicalensis (163–343 mm), S. bungeana (164–355 mm), S. grandis (148–286 mm), and S. breviflora (130–316 mm); severe drought or excessive rainfall would be expected to result in a reduced impact of elevated CO2. Elevated CO2 affected the Nmass of S. grandis only in a narrow precipitation range. The effect of elevated CO2 reached a maximum when the amount of precipitation was 253, 260, 217, and 222 mm for S. baicalensis, S. bungeana, S. grandis, and S. breviflora, respectively. The Nmass of S. grandis was the least sensitive to elevated CO2. The Nmass of S. breviflora was more sensitive to elevated CO2 under a drought condition compared with the other Stipa species. 相似文献
15.
Lennart Hilbert Shivaram Cumarasamy Nedjma?B. Zitouni Michael?C. Mackey Anne-Marie Lauzon 《Biophysical journal》2013,105(6):1466-1474
Naturally occurring groups of muscle myosin behave differently from individual myosins or small groups commonly assayed in vitro. Here, we investigate the emergence of myosin group behavior with increasing myosin group size. Assuming the number of myosin binding sites (N) is proportional to actin length (L) (N = L/35.5 nm), we resolve in vitro motility of actin propelled by skeletal muscle myosin for L = 0.2–3 μm. Three distinct regimes were found: L < 0.3 μm, sliding arrest; 0.3 μm ≤ L ≤ 1 μm, alternation between arrest and continuous sliding; L > 1 μm, continuous sliding. We theoretically investigated the myosin group kinetics with mechanical coupling via actin. We find rapid actin sliding steps driven by power-stroke cascades supported by postpower-stroke myosins, and phases without actin sliding caused by prepower-stroke myosin buildup. The three regimes are explained: N = 8, rare cascades; N = 15, cascade bursts; N = 35, continuous cascading. Two saddle-node bifurcations occur for increasing N (mono → bi → mono-stability), with steady states corresponding to arrest and continuous cascading. The experimentally measured dependence of actin sliding statistics on L and myosin concentration is correctly predicted. 相似文献
16.
Neonicotinoid insecticides are one of the most important commercial insecticides used worldwide. The potential toxicity of the residues present in environment to humans has received considerable attention. In this study, a novel Ochrobactrum sp. strain D-12 capable of using acetamiprid as the sole carbon source as well as energy, nitrogen source for growth was isolated and identified from polluted agricultural soil. Strain D-12 was able to completely degrade acetamiprid with initial concentrations of 0–3000 mg·L−1 within 48 h. Haldane inhibition model was used to fit the special degradation rate at different initial concentrations, and the parameters q
max, K
s and K
i were determined to be 0.6394 (6 h)−1, 50.96 mg·L−1 and 1879 mg·L−1, respectively. The strain was found highly effective in degrading acetamiprid over a wide range of temperatures (25–35°C) and pH (6–8). The effects of co-substrates on the degradation efficiency of acetamiprid were investigated. The results indicated that exogenously supplied glucose and ammonium chloride could slightly enhance the biodegradation efficiency, but even more addition of glucose or ammonium chloride delayed the biodegradation. In addition, one metabolic intermediate identified as N-methyl-(6-chloro-3-pyridyl)methylamine formed during the degradation of acetamiprid mediated by strain D-12 was captured by LC-MS, allowing a degradation pathway for acetamiprid to be proposed. This study suggests the bacterium could be a promising candidate for remediation of environments affected by acetamiprid. 相似文献
17.
18.
Estefanía Sierra-Ibarra Jorge Alcaraz-Cienfuegos Alejandra Vargas-Tah Alberto Rosas-Aburto ngeles Valdivia-Lpez Martín G Hernndez-Luna Eduardo Vivaldo-Lima Alfredo Martinez 《Journal of industrial microbiology & biotechnology》2022,49(2)
Teak wood residues were subjected to thermochemical pretreatment, enzymatic saccharification, and detoxification to obtain syrups with a high concentration of fermentable sugars for ethanol production with the ethanologenic Escherichia coli strain MS04. Teak is a hardwood, and thus a robust deconstructive pretreatment was applied followed by enzymatic saccharification. The resulting syrup contained 60 g l–1 glucose, 18 g l–1 xylose, 6 g l–1 acetate, less than 0.1 g l–1 of total furans, and 12 g l–1 of soluble phenolic compounds (SPCs). This concentration of SPC is toxic to E. coli, and thus two detoxification strategies were assayed: (1) treatment with Coriolopsis gallica laccase followed by addition of activated carbon and (2) overliming with Ca(OH)2. These reduced the phenolic compounds by 40% and 76%, respectively. The detoxified syrups were centrifuged and fermented with E. coli MS04. Cultivation with the overlimed hydrolysate showed a 60% higher volumetric productivity (0.45 gETOH l–1 hr–1). The bioethanol/sugar yield was over 90% in both strategies. 相似文献
19.
Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici 总被引:2,自引:0,他引:2
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Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (Pi). To better understand phosphorus movement between the bacteroid and the host plant, Pi transport was characterized in R. tropici. We observed two Pi transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The Km and Vmax values for the low-affinity system were estimated to be 34 ± 3 μM Pi and 118 ± 8 nmol of Pi · min−1 · mg (dry weight) of cells−1, respectively, and the Km and Vmax values for the high-affinity system were 0.45 ± 0.01 μM Pi and 86 ± 5 nmol of Pi · min−1 · mg (dry weight) of cells−1, respectively. Both systems were inducible by Pi starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but Pi transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the Pi transport rate was correlated with the intracellular ATP concentration. Also, Pi movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both Pi transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity. 相似文献
20.
Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed. 相似文献