StyS激酶自磷酸化对Pseudomonas putida B3中靛蓝生物合成的调节作用

在恶臭假单胞菌Pseudomonas putida B3中,靛蓝生物合成关键酶基因styAB上游存在一个二元调控系统StyS-StyR。StyS蛋白属于激酶家族,是磷酸化信号转导的重要媒介,但激酶StyS的自磷酸化传导机制对靛蓝生物合成的调节作用尚未探明,因此,研究以Pseudomonas putida B3中激酶蛋白StyS作为研究对象,发现了两个磷酸化结合位点,构建了磷酸化位点突变株。通过分析野生菌株P. putida B3、styS基因缺失菌株P. putida B3-ΔstyS、styS基因回补菌株P. putida B3-ΔstyS-8和磷酸化位点突变株P. putida B3-ΔstyS-1之间的靛蓝产量及酶活发现,P. putida B3产量为10.14mg/L,P. putida B3-ΔstyS-8产量为3.63mg/L,磷酸化位点突变菌株无激酶活性且失去了产生靛蓝的能力。结果表明,StyS自磷酸化作用在靛蓝发酵体系中起重要作用。研究结果将为靛蓝生物合成途径的进一步研究提供参考。     

New developments and directions in the area of elastomers and rubberlike elasticity

There are number of important developments in the area of elastomeric polymers, including (i) network chains of controlled stiffness, (ii) model elastomers (including dangling-chain networks), (iii) fluorosiloxane elastomers, (iv) new thermoplastic elastomers, (v) other new elastomers, (v) bimodal network chain-length distributions, (vi) cross linking in solution or in a state of deformation, and (vii) gel collapse. Interesting elastomeric composites include those with (i) in-situ generated ceramic-like particles, (ii) ellipsoidal fillers, (iii) clay-like layered fillers, (iv) polyhedral oligomeric silsesquioxane (POSS) particles, (v) porous fillers, (vi) elastomeric domains modifying ceramics, and (vii) controlled interfaces. New characterization techniques are being developed for elastomers, and there have been new developments in elasticity theory and in elastomer processing. Some examples of societal aspects of relevance are (i) synthesis of elastomers in environmentally-friendly solvents, (ii) biosynthesis, (iii), recyclability, (iv) improved adhesion to tire cords, and (v) better barrier properties in anti-terrorism clothing. Educational topics include curriculum development, and mobile laboratories for elastomer experiments and demonstrations.     

Structural Analysis of Saccharomyces cerevisiae Dihydroorotase Reveals Molecular Insights into the Tetramerization Mechanism

Dihydroorotase (DHOase), a dimetalloenzyme containing a carbamylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase (ALLase), dihydropyrimidinase (DHPase), hydantoinase, and imidase. Unlike most known cyclic amidohydrolases, which are tetrameric, DHOase exists as a monomer or dimer. Here, we report and analyze two crystal structures of the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase) complexed with malate. The structures of different DHOases were also compared. An asymmetric unit of these crystals contained four crystallographically independent ScDHOase monomers. ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). Unlike EcDHOase, ScDHOase can form tetramers, both in the crystalline state and in solution. In addition, the subunit-interacting residues of ScDHOase for dimerization and tetramerization are significantly different from those of other DHOases. The tetramerization pattern of ScDHOase is also different from those of DHPase and ALLase. Based on sequence analysis and structural evidence, we identify two unique helices (α6 and α10) and a loop (loop 7) for tetramerization, and discuss why the residues for tetramerization in ScDHOase are not necessarily conserved among DHOases.     

Mechanisms Affecting the Biosynthesis and Incorporation Rate of Selenocysteine

Selenocysteine (Sec) is the 21st non-standard proteinogenic amino acid. Due to the particularity of the codon encoding Sec, the selenoprotein synthesis needs to be completed by unique mechanisms in specific biological systems. In this paper, the underlying mechanisms for the biosynthesis and incorporation of Sec into selenoprotein were comprehensively reviewed on five aspects: (i) the specific biosynthesis mechanism of Sec and the role of its internal influencing factors (SelA, SelB, SelC, SelD, SPS2 and PSTK); (ii) the elements (SECIS, PSL, SPUR and RF) on mRNA and their functional mechanisms; (iii) the specificity (either translation termination or translation into Sec) of UGA; (iv) the structure–activity relationship and action mechanism of SelA, SelB, SelC and SelD; and (v) the operating mechanism of two key enzyme systems for inorganic selenium source flow before Sec synthesis. Lastly, the size of the translation initiation interval, other action modes of SECIS and effects of REPS (Repetitive Extragenic Palindromic Sequences) that affect the incorporation efficiency of Sec was also discussed to provide scientific basis for the large-scale industrial fermentation for the production of selenoprotein.     

一种生物合成基钻井液在长宁气田的应用

针对页岩气水平井用油基钻井液环境污染性强、废弃物处理难度大等缺点,开展了一种新型的页岩气井用环保型钻井液体系研究。通过生物合成基础油制备和系统配方性能实验评价,研发了一种以改性植物油为连续相,盐水为分散相的油包水乳化钻井液体系;该体系不含芳香烃等组分,易生物降解,废弃物满足环保标准。将生物合成基钻井液在长宁气田HA平台3口井中进行了应用,应用效果表明,该体系抑制性、封堵性和润滑性好,能保障钻井、电测和下套管等作业顺利施工,环保风险和废弃物处理成本低。     

Origin of the C3-unit in placidenes: further insights into taxa divergence of polypropionate biosynthesis in marine molluscs and fungi

Structural diversity of polypropionates arises from the amazing combination of acetate and propionate units. Feeding experiments with single and doubly ¹³C-labelled precursors prove that the Mediterranean slug Placida dendritica utilizes intact C₃-units for the biosynthesis of placidenes (e.g., 1-5), prototypes of a family of pyrone-containing polypropionates largely represented in fungi and marine invertebrates. These results show that fungi and molluscs have elaborated two distinct polyketide pathways for the synthesis of similar or even identical compounds.     

Increasing the accuracy of mass isotopomer analysis through calibration curves constructed using biologically synthesized compounds

Mass isotopomer analysis is an important technique to measure the production and flow of metabolites in living cells, tissues, and organisms. This technique depends on accurate quantifications of different mass isotopomers using mass spectrometry. Constructing calibration curves using standard samples is the most universal approach to convert raw mass spectrometry measurements into quantitative distributions of mass isotopomers. Calibration curve approach has been, however, of very limited use in comprehensive analyses of biological systems, mainly suffering from the lack of extensive range of standard samples with accurately known isotopic enrichment. Here, we present a biological method capable of synthesizing specifically labeled amino acids. These amino acids have well‐determined and estimable mass isotopomer distributions and thus can serve as standard samples. In this method, the bacterium strain Methylobacterium salsuginis sp. nov. was cultivated with partially ¹³C‐labeled methanol as the only carbon source to produce ¹³C‐enriched compounds. We show that the mass isotopomer distributions of the various biosynthesized amino acids are well determined and can be reasonably estimated based on proposed binomial approximation if the labeling state of the biomass reached an isotopic steady state. The interference of intramolecular inhomogeneity of ¹³C isotope abundances caused by biological isotope fractionation was eliminated by estimating average ¹³C isotope abundance. Further, the predictions are tested experimentally by mass spectrometry (MS) spectra of the labeled glycine, alanine, and aspartic acid. Most of the error in mass spectrometry measurements was less than 0.74 mol% in the test case, significantly reduced as compared with uncalibrated results, and this error is expected to be less than 0.4 mol% in real experiment as revealed by theoretical analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural Basis and Enzymatic Mechanism of the Biosynthesis of C9‐ from C10‐Monoterpenoid Indole Alkaloids

Cutting carbons : The three‐dimensional structure of polyneuridine aldehyde esterase (PNAE) gives insight into the enzymatic mechanism of the biosynthesis of C₉‐ from C₁₀‐monoterpenoid indole alkaloids (see scheme). PNAE is a very substrate‐specific serine esterase. It harbors the catalytic triad S87‐D216‐H244, and is a new member of the α/β‐fold hydrolase superfamily. Its novel function leads to the diversification of alkaloid structures.