Production of α-keto acids: 2. Immobilized whole cells of Providencia sp. PCM 1298 containing l-amino acid oxidase

A number of bacteria belonging to the genera Proteus, Providencia, Pseudomonas and Erwinia have been tested for their capacity to oxidize l-amino acids to their corresponding α-keto acids. Members of the Proteus and the Providencia genera were active towards various l-amino acids. Immobilized cell preparations of Providencia sp. PCM 1298 were shown to form up to 80 mg α-keto-γ-methiol butyric acid from l-methionine per g of gel preparation (containing 4% w/w cells) per day. The productivity was highly dependent on the size of the beads. Oxygen appeared to be the rate-limiting substrate and oxygen transfer rates of 3–4 μmol cm^?2h^?1were calculated. The entrapment of activated charcoal to remove H₂O₂ formed during the oxidation extended the half-life of the immobilized biocatalyst considerably. A decrease in l-amino acid oxidase [l-amino acid: oxygen oxidoreductase (deaminating); EC 1.4.3.2] activity during operation could be compensated for by reinoculation of the alginate-entrapped cells in fresh growth medium, allowing use of these preparations of immobilized bacterial cells for more than one month.     

Ectomycorrhizal fungi: A new source of atmospheric methyl halides?

Incomplete source budgets for methyl halides – compounds that release inorganic chlorine and bromine radicals which, in turn, catalyze atmospheric ozone depletion – limit our ability to predict the fate of the stratospheric ozone layer. We report here the first measured emissions of methyl chloride, methyl bromide, and methyl iodide from ectomycorrhizal fungi. We grew nine fungal isolates on growth media containing halide concentrations similar to those found in soils and plant tissues. The observed range of emissions was 0.003–65 μg methyl chloride, 0.001–3 μg methyl bromide, and 0.02–12 μg methyl iodide g^?1 dry weight fungi day^?1. Species varied in production rates of methyl chloride vs. methyl bromide vs. methyl iodide. Cenococcum geophilum, a widespread ectomycorrhizal fungus, was further tested to investigate the effects of halide substrate concentration in growth media. Emissions from this species increased linearly with increasing concentrations of both bromide and iodide. In addition, a subset of four fungi was studied with two media concentrations each of chloride, bromide, and iodide (0.2 or 20 mm ). These fungi had similar responses to halide concentration, despite 1000‐fold differences in baseline emission rates between isolates. Finally, high chloride concentrations (20 mm ) in media did not appear to inhibit emissions of methyl bromide or methyl iodide. Overall, ectomycorrhizal fungi might be an important source of methyl halides to the atmosphere, and substrate concentrations and community composition may influence production levels in ecosystems.     

The effect of three α-keto acids on 3-mercaptopyruvate sulfurtransferase activity

3-Mercaptopyruvate sulfurtransferase catalyzes the transfer of sulfur from 3-mercaptopyruvate to several possible acceptor molecules, one of which is cyanide. Because the transsulfuration of cyanide is the primary in vivo mechanism of detoxification, 3-mercaptopyruvate sulfurtransferase may function in the enzymatic detoxification of cyanide in vivo. Three α-keto acids (α-ketobutyrate, α-ketoglutarate, and pyruvate) have previously been demonstrated to be cyanide antidotes in vivo, and it has been suggested that this is due to the nonenzymatic binding of cyanide by the α-keto acid. However, it has also been proposed that α-keto acids may increase the activity of enzymes involved in the transsulfuration of cyanide. Thus, the effect of these three α-keto acids on the enzyme 3-mercaptopyruvate sulfurtransferase was examined. All three α-keto acids inhibited 3-mercaptopyruvate sulfurtransferase in a concentration-dependent manner and were determined to be uncompetitive inhibitors of MST with respect to 3-mercaptopyruvate. The inhibitor constant K_i was estimated by two methods for each inhibitor and ranged from 4.3 to 6.3 mM. The I₅₀, which is the inhibitor concentration that produces 50% inhibition, was calculated for all three α-keto acids and ranged between 9.5 and 13.7 mM. These observations add further support to the hypothesis that the mechanisms of the α-keto acid antidotes is the nonenzymatic binding of cyanide, not stimulation of enzymes involved in the transsulfuration of cyanide to thiocyanate. © 1996 John Wiley & Sons, Inc.     

Methyl chloride isotopic signatures from Irish forest soils and a comparison between abiotic and biogenic methyl halide soil fluxes

Forest soils demonstrate in a microcosm the difficulties that are faced in quantifying methyl halide budgets. Carbon isotopic analyses have been proposed as a potential tool to address these concerns and in this study we have measured significant enrichment of the methyl chloride ¹³C/¹²C isotopic ratio (from ?40.2 ± 0.8‰ to ?33.4 ± 7.4‰) after 9 min chamber emplacement on local Irish forest soils. This enrichment occurred independent of direction of methyl chloride fluxes. Measurements from soil cores in a flow‐through system (FTS) are comparable with chamber‐based isotopic measurements and indicate that methyl chloride produced abiotically from organic soil horizons has an isotopic ¹³C signature of ?53 ± 49‰, significantly less depleted than previously reported. Average net methyl chloride, methyl bromide and methyl iodide fluxes from soils (77.8 ± 2.1, 1.25 ± 3.63 and 0.35 ± 2.00 μg MeX m^?2 day^?1, respectively) are in line with previously reported values; however, a better understanding of spatial and temporal variability is needed for budget quantification. Methyl halide fluxes from FTS soil cores demonstrate that, on a per gram basis, most consumption occurs through biologically driven processes in the O horizon, with progressively smaller contributions in deeper horizons. Sporadic biogenic production was observed in shallow soil horizons only. Abiotic production was at most one‐tenth the net biological reaction rate in the O horizon and did not appear to be significantly different from zero in lower horizons. Modelled emissions based upon observed and reported rates for production, consumption and diffusion within the soil atmosphere system are unable to replicate all observed isotopic signatures from chamber fluxes.     

3-Ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids

The various neutral amino acids and aliphatic 2-keto acids exhibit differential effects on insulin secretion. The common denominator for all these effects is the 2-ketoglutarate generation in the pancreatic B-cell mitochondria. The neutral amino acidsl-leucine andl-norvaline and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, 2-ketovalerate, and 2-keto-3-methylvalerate all stimulate insulin secretion and increase 2-ketoglutarate generation in pancreatic B-cell mitochondria through activation of glutamate dehydrogenase and transamination withl-glutamate andl-glutamine, respectively. The neutral amino acidsl-valine,l-norleucine, andl-alanine and the aliphatic 2-keto acids 2-ketoisovalerate and pyruvate do not stimulate insulin secretion and do not increase 2-ketoglutarate generation in pancreatic B-cell mitochondria. Inhibition of 2-keto acid induced insulin secretion byl-valine andl-isoleucine is accompanied by reduced 2-ketoglutarate generation in pancreatic B-cell mitochondria. Thus intramitochondrial 2-ketoglutarate generation in pancreatic B-cells may regulate the insulin secretory potency of amino acids and 2-keto acids.     

Effect of deuteration on some structural parameters of methyl groups in proteins as evaluated by residual dipolar couplings

One bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar and residual dipolar couplings have been measured at sites in an ¹⁵N, ¹³C, 50% ²H labeled sample of the B1 immunoglobulin binding domain of peptostreptococcal protein L to investigate changes in the structure of methyl groups in response to deuterium substitution. Both one bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar coupling constants have been found to decrease slightly with increasing deuterium content. Previous studies have shown that ¹H-¹³C couplings in methyl groups are exquisitely sensitive to electronic structure, with decreases in coupling values as a function of deuteration consistent with a slight lengthening of the remaining H-C bonds. Changes in the H_methylC_methylC angle are found to be small, with average differences on the order of 0.3 ± 0.1° and 0.4 ± 0.2° between CH₃, CH₂D and CH₃, CHD₂ isotopomers, respectively. Knowledge of methyl geometry is a prerequisite for the extraction of accurate dynamics parameters from spin relaxation studies involving these groups.     

Metabolic engineering of Escherichia coli for production of fatty acid short-chain esters through combination of the fatty acid and 2-keto acid pathways

Fatty acid short-chain esters (FASEs) are biodiesels that are renewable, nontoxic, and biodegradable biofuels. A novel approach for the biosynthesis of FASEs has been developed using metabolically-engineered E. coli through combination of the fatty acid and 2-keto acid pathways. Several genetic engineering strategies were also developed to increase fatty acyl-CoA availability to improve FASEs production. Fed-batch cultivation of the engineered E. coli resulted in a titer of 1008 mg/L FASEs. Since the fatty acid and 2-keto acid pathways are native microbial synthesis pathways, this strategy can be implemented in a variety of microorganisms to produce various FASEs from cheap and readily-available, renewable, raw materials such as sugars and cellulose in the future.     

开同对维持性血液透析患者营养及脂质代谢的影响

目的：研究复方α-酮酸对维持性血液透析（MHD）患者营养及脂质代谢的影响。方法：将40例MHD患者随机分为两组：（1）实验组（n=20）,予常规蛋白饮食,并服用复方α-酮酸12片／d;（2）对照组（n=20）,子常规蛋白饮食,不服用复方α-酮酸。分别于治疗前、治疗后3个月后测定各项血生化指标及人体测量学指标。结果：经3个月治疗后,实验组血白蛋白、前白蛋白较前明显升高（P〈0．01）,总胆固醇和甘油三酯明显下降（P〈0．01）,血红蛋白无明显变化（P〉0．05）;对照组血红蛋白、血白蛋白、前白蛋白及血脂无明显变化;实验组及对照组治疗前后体重指数、肱三头肌皮褶厚度、上臂肌围无明显变化（P〉0．05）。结论：复方α-酮酸可有效改善维持性血液透析患者营养状况以及脂质代谢,但长期使用该疗法的疗效和安全性有待于进一步的研究。     

Molecular mechanisms underlying Grateloupia imbricata (Rhodophyta) carposporogenesis induced by methyl jasmonate

When applied in vitro, methyl jasmonate is sensed by the red seaweed Grateloupia imbricate, substantially and visually affecting its carposporogenesis. However, although there is some understanding of the morphological changes induced by methyl jasmonate in vitro, little is known about the genes that are involved in red seaweed carposporogenesis and how their protein products act. For the work reported herein, the expression of genes in red seaweed that encode enzymes involved in the synthesis of methyl jasmonate (jasmonic acid carboxyl methyl transferase and a putative methyl transferase) was monitored. Additionally the genes involved in oxidation (cytochrome P450 and WD40), jasmonate synthesis, signal transduction, and regulation of reactive oxygen species (MYB), and reproduction (ornithine decarboxylase) were monitored. To determine when or if the aforementioned genes were expressed during cystocarp development, fertilized and fertile thalli were exposed to methyl jasmonate and gene expression was measured after 24 and 48 h. The results showed that methyl jasmonate promoted differential gene expression in fertilized thalli by 24 h and upregulated expression of the ornithine decarboxylase gene only by 48 h in fertile thalli (0.75 ± 003 copies · μL^?1 at 24 h vs. 1.11 ± 0.04 copies · μL^?1 at 48 h). We conclude that Ornithine decarboxylase expression involves methyl jasmonate signaling as well as development and maturation of cystocarps.     

Preparation of Enzymes Required for Enzymatic Quantification of 5-Keto-D-gluconate and 2-Keto-D-gluconate

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.     

Heterocyclic β-keto sulfide derivatives of carvacrol: Synthesis and copper (II) ion reducing capacity

Sixteen β-keto sulfide derivatives of carvacrol (4–19) incorporating phenyl or N, O and S heterocyclic moieties were synthesized in three steps. The relationships between heterocyclic structure and cupric, Cu(II), ion reducing antioxidant capacity (CUPRAC) were examined. Nine of the compounds (8–9 and 13–19) showed better CUPRAC activity than trolox at neutral pH, with trolox equivalent antioxidant capacity (TEAC) coefficients ranging between 1.20 and 1.75. Two derivatives (11–12) showed comparable reducing capacity to trolox, with TEAC values of 0.95 for 11 and 1.02 for 12. Compounds 8–9 and 11–19 were more effective at reducing the Cu(II) ion than ascorbic acid and the parent compound, carvacrol. The most effective antioxidants were those containing an oxadiazole, thiadiazole or triazole moiety. In particular, the methyl thiadiazole derivative (15) had the highest Cu(II) ion reducing capacity, with a TEAC coefficient of 1.73.     

An easy method for preparing radioactive methyl esters of eicosanoids suitable as ligands in radioimmunoassays

A rapid and convenient method is described for methylating prostanoids and other arachidonic acid metabolites. With ³H-methyl iodide of high specific activity, tracers for radioimmunoassay can be produced at a cost which is only a fraction of that of labeled compounds currently available. In radioimmunoassays for PGE₂, TXB₂ and 6-keto -PGF_1α, labeled methyl esters gave results which were comparable to those obtained with the use of tritiated free acids as radioligands.     

Measurement of methyl 13C-1H cross-correlation in uniformly 13C-, 15N-, labeled proteins

An understanding of side chain motions in protein is of great interest since side chains often play an important role in protein folding and intermolecular interactions. A novel method for measuring the dynamics of methyl groups in uniformly ¹³C-, ¹⁵N-labeled proteins has been developed by our group. The method relies on the difference in peak intensities of ¹³C quartet components of methyl groups, in a spectrum recording the free evolution of ¹³C under proton coupling in a constant-time period. Cross-correlated relaxation rates between ¹³C-¹H dipoles can be easily measured from the intensities of the multiplet components. The degree of the methyl restrictions (S ²) can be estimated from the cross-correlated relaxation rate. The method is demonstrated on a sample of human fatty acid binding protein in the absence of fatty acid. We obtained relaxation data for 33 out of 46 residues having methyl groups in apo-IFABP. It has been found that the magnitude of the CSA tensor of spin ¹³C in a methyl group could be estimated from the intensities of the ¹³C multiplet components.     

Estimates of methyl 13C and 1H CSA values (Δσ) in proteins from cross-correlated spin relaxation

Simple pulse schemes are presented for the measurement of methyl ¹³C and ¹H CSA values from ¹H–¹³C dipole/¹³C CSA and ¹H–¹³C dipole/¹H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average ¹³C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged ¹H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group ¹H CSA in dimethylmalonic acid.     

Origin of the methyl carbon of methyl coniine in Conium maculatum

Studies with [methyl-¹⁴C]-l-methionine have established that the methyl carbon of l-methionine can act as a precursor of the N-methyl group of methyl coniine in Conium maculatum.     

Electrophysiological evidence for detection and discrimination of pheromonal bile acids by the olfactory epithelium of female sea lampreys (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

Electro-olfactograms were used to determine sensitivity and specificity of olfactory organs of female sea lampreys (Petromyzon marinus) to four bile acids: 3-keto petromyzonol sulfate and 3-keto allocholic acid from spermiating males and petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are thought to function as a mating pheromone and larval bile acids as a migratory pheromone. The response threshold was 10^–12 mol l^–1 for 3-keto petromyzonol sulfate and 10^–10 mol l^–1 for the other bile acids. At concentrations above 10^–9 mol l^–1, the sulfated bile acids showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol sulfate and water conditioned with spermiating males induced similar concentration-response curves and response thresholds. Cross-adaptation experiments demonstrated that the sulfated and non-sulfated bile acids represent different odors to the olfactory epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and allocholic acid represent the same odor. Results indicate that male-specific bile acids are potent and specific stimulants to the female olfactory organ, supporting the previous hypothesis that these bile acids function as a pheromone.Abbreviations 3kACA 3-keto allocholic acid - 3kPZS 3-keto petromyzonol sulfate - ACA allocholic acid - ANOVA analysis of variance - ELISA enzyme-linked immunosorbent assay - EOG electro-olfactogram - PIR percent initial response - PZS petromyzonol sulfate - SMW spermiating male washings     

Synthesis and properties of methyl 9,12,15-octadecatrienoate geometric isomers

The eight geometrically isomeric methyl 9,12,15-octadecatrienoates were prepared by using the Wittig reaction to couple cis- or trans-3-hexyenyltriphenylphosphonium bromide and methyl 12-oxo-cis- or trans-9-dodecenoate. Pairs of geometric triene isomers formed were separated by partial silver resin chromatography. Physical constants including melting points, percent trans by infrared, equivalent chain lengths (ECL), and ¹³C nuclear magnetic resonance (NMR) chemcial shifts are tabulated for the individual isomers.     

Multiplet component separation for measurement of methyl 13C-1H dipolar couplings in weakly aligned proteins

A simple spectral editing procedure is described that generates separate subspectra for the methyl ¹³C-¹H₃ multiplet components of ¹H-¹³C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields ¹H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of ¹H-¹³C J and dipolar couplings. The editing procedure is illustrated for a Ca²⁺-calmodulin sample in isotropic and liquid crystalline phases.     

Effect of azoxystrobin and kresoxim‐methyl on rice blast and rice grain yield in China

Sensitivity to azoxystrobin and kresoxim‐methyl of 80 single‐spore isolates of Magnaporthe oryzae was determined. The EC₅₀ values for azoxystrobin and kresoxim‐methyl in inhibiting mycelial growth of the 80 M. oryzae isolates were 0.006–0.056 and 0.024–0.287 µg mL^?1, respectively. The EC₅₀ values for azoxystrobin and kresoxim‐methyl in inhibiting conidial germination of the M. oryzae populations were 0.004–0.051 and 0.012–0.105 µg mL^?1, respectively. There was significant difference in sensitivity to azoxystrobin or kresoxim‐methyl between the tested isolates representing differential sensitivity to carbendazim (MBC) and kitazin P (IBP); however, there was no correlation between this difference in sensitivity to azoxystrobin or kresoxim‐methyl and sensitivity to MBC or IBP, indicating that there was no cross‐resistance between azoxystrobin or kresoxim‐methyl and MBC or IBP. In the protective and curative experiments, kresoxim‐methyl exhibited higher protective and curative activity than azoxystrobin when applied at 150 and 250 µg mL^?1 accordingly, while azoxystrobin exhibited stronger inhibitory activity against M. oryzae isolates than that of kresoxim‐methyl in the in vitro test. The results of field experiments also suggested that both azoxystrobin and kresoxim‐methyl at 187.5 g.a.i. ha^?1 gave over 73% control efficacy in both sites, exhibiting excellent activity against rice blast. Taken together, azoxystrobin and kresoxim‐methyl could be a good substitute for MBC or IBP for controlling rice blast in China, but should be carefully used as they were both at‐risk.     

Na+-dependent methyl β-thiogalactoside transport in Salmonella typhimurium

We have studied the role of sodium ions in methyl β-thiogalactoside (TMG) transport via the melibiose permease (TMG II) in SalmonellaTMG uptake via TMG Il in anaerobic, starved and metabolically poisoned cells is dependent on an inward-directed Na⁺ gradient.Cells which have been partially depleted of endogenous substrates show H⁺ extrusion upon sodium-stimulated TMG influx.Measurements of the electrochemical H⁺ gradient in cells, starved in different ways for endogenous substrates, suggest that this proton extrusion is probably not linked to the actual translocation mechanism but is the result of metabolism induced by TMG plus Na⁺ uptake.