High-resolution solution structure of Bacillus subtilis IIAglc

The high-resolution solution structure of the phosphocarrier protein IIA^glc from Bacillus subtilis is determined using 3D and 4D heteronuclear NMR methods. B. subtilis IIA^glc contains 162 amino acid residues and is one of the larger proteins for which high-resolution solution structure has been determined by NMR methods. The structures have been calculated from a total of 2,232 conformational constraints. Comparison with the X-ray crystal structure indicates that the overall fold is the same in solution and in crystalline environments, although some local structural differences are observed. These occur largely in turns and loops, and mostly correspond to regions with high-temperature factors in the crystal structure. The N-terminus of IIA^glc is disordered in solution. The active site is located in a concave region of the protein surface. The histidine, which accepts the phosphoryl group (His 83), interacts with a neighboring histidine (His 68) and is surrounded by hydrophobic residues. Proteins 31:258–270, 1998. © 1998 Wiley-Liss, Inc.     

Requirements for the phosphorylation of the Escherichia coli EIIANtr protein in vivo

The nitrogen-related phosphotransferase system (Ntr-PTS) is a paralogous system working in parallel to the well-known carbohydrate:PTS. In a chain of phosphotransfer reactions, EI^Ntr and NPr (PtsO) deliver phosphoryl groups to the EIIA^Ntr (PtsN) protein. EIIA^Ntr is implicated in important regulatory processes such as the σ^E-dependent cell envelope stress response and regulation of K⁺ uptake. Phosphorylation is believed to trigger the output of EIIA^Ntr in these regulations. EIIA^Ntr is encoded within the gene cluster ptsN–yhbJ–ptsO , which is highly conserved in Proteobacteria . In this study, we investigated the phosphorylation of the Escherichia coli EIIA^Ntr protein in vivo by ³²P-labeling. We show that EIIA^Ntr is readily phosphorylated in wild-type cells. This phosphorylation occurs at a single site, the histidine 73 in EIIA^Ntr. YhbJ and NPr are dispensable for this phosphorylation. A detailed analysis revealed that both the energy coupling phosphotransferases of the Ntr-PTS as well as the 'sugar'-PTS contribute to the phosphorylation of EIIA^Ntr, suggesting cross talk between both systems.     

Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

Synthesis and reactivity of nonstabilized diazo sugars

1,6-Anhydro-4-deoxy-4-diazo-2,3-O-isopropylidene-beta-D-lyxo-hexopyranose (4) is a stable crystalline compound readily accessible by an improved synthetic procedure. It has been used as a model for evaluating the reactivity of the diazo group, when not stabilized by an adjacent carbonyl function, in a rigid chiral matrix. A range of carbene-type, electrophile-promoted, and 1,3-dipolar reactions were evaluated, leading to 4,4'-alkene dimers, 4-deoxy-3-enose and related derivatives, 4,4-dihalo compounds, 4-spirocyclopropane derivatives, 4-spiropyrazole structures, and by skeletal rearrangement, branched-chain anhydropentose structures having a bicyclo[2.2.2] skeleton.     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

Functional interaction between information processing pathways in Escherichia coli and the nature of Δ-H+ (redox)-sensing

Glucose taxis and O₂-taxis in Escherichia coli signal to flagella via a pathway that includes PTS^glc and adenylate cyclase. Information from a number of attractants and repellents is focused at the level of methy-accepting chemotaxis proteins (MCPs) and information is passed to flagella by a separate pathway. Mutants defective in adenylate cyclase (Δcya) had a residual taxis to glucose that was eliminated by preincubating the cells with MCP attractants, or by depleting the -CH₃ donor. A methyltransferase mutant had a decreased sensitivity to MCP repellents and this response was completely blocked by preincubating the cells with glucose. Likewise, the response of cells, depleted for -CH₃, towards repellents, was blocked if bacteria carried a pts mutation. It is concluded that PTS and MCP pathways exchange information. In cya cells, O₂ taxis was restored in the presence of maltose, an MCPII attractat. It is suggested that MCPII is an additional protonmotive force (pmf) sensor.     

霍乱弧菌甘露醇PTS操纵子中mtlR为转录抑制基因

产毒和非产毒的El Tor生物型霍乱弧菌对甘露醇发酵利用的速率有明显差别,在霍乱致病株的快速判断中有重要的参考价值。通过比较快发酵菌株(非产毒株)和慢发酵菌株(产毒株)mtlR缺失突变株与野生株在含0.2%甘露醇的M9培养液及甘露醇发酵液中生长、产酸等的变化,定性地证明了mtlR基因的抑制作用;另外通过定量RT-PCR进一步验证了MtlR蛋白在mtlCBA转录水平发挥负调控作用。但是mtlR还不是引起快慢发酵菌株对甘露醇发酵差异的直接原因。本研究也为我们研究霍乱弧菌甘露醇快慢发酵差异机制提供了必要的参考依据。     

Production of itaconic acid from pentose sugars by Aspergillus terreus

Itaconic acid (IA), an unsaturated 5‐carbon dicarboxylic acid, is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has potential to serve as low‐cost source of sugars for production of IA. Research needs to be performed to find a suitable A. terreus strain that can use lignocellulose‐derived pentose sugars and produce IA. One hundred A. terreus strains were evaluated for the first time for production of IA from xylose and arabinose. Twenty strains showed good production of IA from the sugars. Among these, six strains (NRRL strains 1960, 1961, 1962, 1972, 66125, and DSM 23081) were selected for further study. One of these strains NRRL 1961 produced 49.8 ± 0.3, 38.9 ± 0.8, 34.8 ± 0.9, and 33.2 ± 2.4 g IA from 80 g glucose, xylose, arabinose and their mixture (1:1:1), respectively, per L at initial pH 3.1 and 33°C. This is the first report on the production of IA from arabinose and mixed sugar of glucose, xylose, and arabinose by A. terreus. The results presented in the article will be very useful in developing a process technology for production of IA from lignocellulosic feedstocks. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1059–1067, 2017     

四膜虫：毒理学与生态毒理学研究中的优良模式生物

四膜虫 (Tetrahymena)是毒理学与生态毒理学研究的良好模式生物之一。本文回顾了近年来以四膜虫为实验生物开展的毒理学与生态毒理学研究进展 ,并针对目前产生严重环境压力的持久性有毒化学污染物 ,提出了以四膜虫为模式生物开展相应的生态毒理学研究的前景以及可行的方法。     

Molecular basis of mitochondrial DNA disease

Mitochondrial ATP production via oxidative phosphorylation (OXPHOS) is essential for normal function and maintenance of human organ systems. Since OXPHOS biogenesis depends on both nuclear- and mitochondrial-encoded gene products, mutations in both genomes can result in impaired electron transport and ATP synthesis, thus causing tissue dysfunction and, ultimately, human disease. Over 30 mitochondrial DNA (mtDNA) point mutations and over 100mtDNA rearrangements have now been identified as etiological factors in human disease. Because of the unique characteristics of mtDNA genetics, genotype/phenotype associations are often complex and disease expression can be influenced by a number of factors, including the presence of nuclear modifying or susceptibility alleles. Accordingly, these mutations result in an extraordinarily broad spectrum of clinical phenotypes ranging from systemic, lethal pediatric disease to late-onset, tissue-specific neurodegenerative disorders. In spite of its complexity, an understanding of the molecular basis of mitochondrial DNA disease will be essential as the first step toward rationale and permanent curative therapy.     

On the regioselectivity of the Hanessian-Hullar reaction in 4,6-O-benzylidene protected galactopyranosides

The N-bromosuccinimide mediated fragmentation of methyl 4,6-O-benzylidene-beta-D-galactopyranoside results in the formation of methyl 4-O-benzoyl-6-bromo-6-deoxy-beta-D-galactopyranoside and methyl 4-O-benzoyl-3-bromo-3-deoxy-beta-D-gulopyranoside, as opposed to the methyl 6-O-benzoyl-3-bromo-3-deoxy-beta-D-gulopyranoside originally reported. The kinetic methyl 4-O-benzoyl-6-bromo-6-deoxy-beta-D-galactopyranoside rearranges to the thermodynamic methyl 4-O-benzoyl-3-bromo-3-deoxy-beta-D-gulopyranoside under the reaction conditions, likely via a 3,6-anhydro galactopyranoside. The NBS-mediated cleavage of 4,6-O-benzylidene acetals in the galactopyranoside series is therefore shown to conform to the regiochemistry observed in the corresponding gluco- and mannopyranoside series with preferential cleavage of the C6-O6 bond by an ionic mechanism.     

Synthesis of 2,3- or 1,2-unsaturated derivatives of 2-deoxy-2-trifluoromethylhexopyranoses

The attempted conversion, by treatment with CsF/TBFA in MeCN, of acetylated derivatives of 2-chlorodifluoromethyl-2-deoxyhexopyranoses into their corresponding 2-trifluoromethyl derivatives was always accompanied by an elimination reaction. Thus, representative educts with the D-gluco- and D-manno-configuration gave derivatives of 2,3-dideoxy-2-trifluoromethyl-D-erythro-hex-2-enopyranose and 1,5-anhydro-2-deoxy-2-trifluoromethyl-d-arabino-hex-1-enitol, respectively. X-ray analyses are given for 1,3,4,6-tetra-O-acetyl-2-chlorodifluoromethyl-2-deoxy-alpha-D-mannopyranose and 4,6-di-O-acetyl-2,3-dideoxy-2-trifluoromethyl-alpha-D-erythro-hex-2-enopyranose.