Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

肝素C5异构酶的表达优化及分子改造

肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase,C5,EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶,催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5号位羧基翻转生成L-艾杜糖醛酸 (L-iduronic acid,L-IdoA)。文中以大肠杆菌Escherichia coli为宿主对斑马鱼来源的肝素葡萄糖醛酸C5异构酶基因Glce进行重组表达优化与分子改造。比较了3种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5表达的差异情况,其中以嗜冷启动型载体pCold Ⅲ表达酶活最高,达到(1 873.61±5.42) U/L。为了进一步提高C5的可溶表达量,在N端融合促溶标签SET2后,可溶蛋白表达量比对照提高了50%,酶活达到 (2 409.25±6.43) U/L。在此基础上,通过理性设计对底物结合口袋进行定点突变,获得最优突变体 (V153R) 的酶活和比酶活分别为 (5 804.32±5.63) U/L和(145.14±2.33) U/mg,是原始酶的2.41倍和2.28倍。肝素C5异构酶改造与表达优化为酶法催化合成肝素奠定了基础。     

定点突变提高解淀粉芽孢杆菌JP-21脲酶应用特性

氨基甲酸乙酯(Ethylcarbamate,EC)是一种存在于发酵食品和酒精饮料中的可致癌物,过量摄入可能会影响人体健康。酶法降解是减少发酵食品中氨基甲酸乙酯及其前体尿素含量的有效方法之一。脲酶具有氨基甲酸乙酯水解酶和尿素酶两种活性,因此在减少发酵食品中氨基甲酸乙酯及其前体尿素方面具有良好的应用前景。目前脲酶降解发酵酒精饮料中氨基甲酸乙酯面临的主要问题是脲酶对氨基甲酸乙酯的催化活性及亲和力较低,因而其降解效果不理想。文中成功在大肠杆菌Escherichia coli中表达了来源于解淀粉芽孢杆菌Bacillus amyloliquefaciens JP-21的脲酶,表达水平为尿素酶3 291.74 U/L,氨基甲酸乙酯水解酶227.26 U/L。通过模拟脲酶中催化亚基UreC与氨基甲酸乙酯对接的结构,确定了M326和M374这两个影响酶与底物结合的位点。采用点饱和突变获得了3株氨基甲酸乙酯水解酶活性提高的突变体M374A、M374T和M326V,以EC为底物时的Km分别为101.84mmol/L、129.49 mmol/L和121.67 mmol/L,比野生型分别降低了37.47%–50.82%。突变体可以降解黄酒中97%的尿素,M374T对黄酒中EC的降解效果最好,可将黄酒中EC从513.90μg/L降至393.57μg/L,降解率是野生脲酶的1.97倍。研究结果对今后改造脲酶催化特性和改善其应用特性具有重要意义,可为开发减控发酵食品中的微生物代谢氨(胺)类危害物策略提供参考。     

果胶甲酯酶的结构与功能研究进展

果胶甲酯酶(PME)是一种重要的果胶酶,其水解果胶中的甲酯基从而释放甲醇并降低果胶的甲酯化程度。目前在食品加工、茶饮料、造纸等生产工艺中有着广泛的应用前景。随着对PME的深入研究,已报道了几种不同来源的酶晶体结构,对这些已获得的晶体结构进行分析发现,PME属于右手平行β-螺旋结构,其催化残基为2个保守的天冬氨酸和1个谷氨酰胺残基,并且在催化过程中分别起到了一般酸碱、亲核试剂以及稳定中间体的作用。同时对其底物特异性进行分析,初步了解其底物与活性位点的识别机制。文中针对这几个相关方面进行了系统的综述。     

不同植物来源可溶性有机质对亚热带森林土壤酶活性的影响

探究不同植物来源可溶性有机质(DOM)进入土壤后对酶活性的影响, 可以为降水淋溶下亚热带地区不同森林生态系统土壤碳循环提供科学依据。该研究提取杉木(Cunninghamia lanceolata)、木荷(Schima superba)和楠木(Phoebe zherman) 3种植物鲜叶中的DOM分别输入杉木人工林土壤中, 以等量的去离子水添加为对照, 进行25天的室内培养。培养结束后测定土壤理化性质、微生物生物量和酶活性等指标。结果表明: 与对照处理(CT)相比, 添加3种叶片DOM后, 土壤总有机碳(SOC)、总氮(TN)含量和碳氮比均无显著变化。杉木叶片DOM添加处理(CL)的TN含量显著低于木荷叶片DOM添加处理(SL)和楠木叶片DOM添加处理(PL), 碳氮比显著高于SL和PL。3种叶片DOM输入整体上提高了土壤溶解有机碳(DOC)和溶解有机氮(DON)的含量。叶片DOM输入后土壤微生物生物量碳(MBC)含量无显著变化, 然而CL和SL的土壤微生物生物量氮(MBN)含量分别比CT降低了50.9%和51.1%, PL的MBN含量比CT提高了54.0%。与CT相比, 不同植物来源DOM输入后, β-葡萄糖苷酶(βG)、纤维素水解酶(CBH)和过氧化物酶(PEO) 3种酶活性均显著上升, 而多酚氧化酶(PPO)活性则显著下降; 此外, βG和CBH活性均表现出CL > SL > PL的特征。相关性分析的结果表明, 添加叶片DOM 3种处理的SOC、TN、MBN含量和βG、CBH活性都与所输入DOM的DOC含量和腐殖化指数(HIX)显著相关, 此外, 土壤MBN含量和PPO活性与输入叶片DOM的pH呈正相关关系。冗余分析(RDA)结果表明, 叶片DOM输入后引起土壤酶活性变化的关键因子是DON和DOC含量。总体来说, 不同植物来源DOM性质的差异会影响土壤碳循环水解酶的活性, 而叶片DOM输入后增加了土壤碳和氮的有效性, 引起4种碳循环酶的不同响应。     

不同缓释过氧化钙对潜育环境下水稻土微生物特性的影响

为了解不同缓释过氧化钙对潜育化稻田土壤微生物特性的影响,于网室内进行潜育化稻田环境模拟试验,以不施过氧化钙为对照,探究过氧化钙粉末、过氧化钙颗粒与4种不同释氧效果的包膜过氧化钙在早稻分蘖期同等时期对潜育化稻田土壤活性有机碳、有效养分及微生物特性的影响。结果表明: 施用过氧化钙均能提高土壤活性有机碳、有效养分、微生物生物量及可培养微生物数量和酶活性,包膜过氧化钙对土壤微生物和酶活性的改善效果较好,其次依次为过氧化钙颗粒和过氧化钙粉末。包膜过氧化钙处理中,以乙基纤维素包膜效果最好,与不施过氧化钙处理相比,其土壤活性有机碳、微生物生物量碳、微生物生物量氮、微生物生物量磷分别显著提高19.4%、11.4%、121.5%、127.2%,土壤碱解氮和有效磷分别提高4.0%和45.5%;土壤可培养细菌和可培养微生物总量分别显著提高137.3%和113.7%,真菌和放线菌数量分别提高33.6%和44.7%;蔗糖酶、磷酸酶和脲酶活性分别显著提高92.4%、91.8%和112.5%,过氧化氢酶活性提高17.1%。研究结果可为包膜过氧化钙对潜育化稻田改良提供参考。     

The effect of grazing on winter survival of midday gerbil (Meriones meridianus) of different genders

The objective of this study was to investigate the effects of grazing on midday gerbil (Meriones meridianus) population characteristics and survival of animals of different genders. The experiment used a randomized complete block design and was conducted in Alxa Left Banner, Inner Mongolia, China, in 2002 (The agricultural reclamation plots set up in 1994). From April 2006 to October 2010, midday gerbils were live‐trapped in 3 light grazing plots, 3 overgrazed plots, and 3 grazing exclusion plots. The quantity of vegetation was investigated in the two different grazing intensity areas and grazing exclusion area to determine the relationship between gerbils and plant food availability. The results suggested that there was higher gerbil density, individual body mass, and daily body mass growth rate in the grazing exclusion sites than the other sites across the whole year. Females had higher survival in grazing exclusion areas than in other treatments, but the males’ survival showed the opposite pattern. Our results indicated that grazing negatively influenced the midday gerbil population by reducing food availability. Grazing influenced the survival rates of male midday gerbils positively, but had negative effects on females. The reason for gendered differences in survival rates of midday gerbils requires further investigation.     

Trophic ecology of an introduced top predator (Felis catus) on a small African oceanic islet (Santa Luzia,Cabo Verde Islands)

Studies on feral cat diet offer important ecological information and are the first step towards determining their impact upon endangered species. However, in comparing seasonal changes in diet with seasonal prey availability, the scarce amount of research into oceanic islands worldwide must be considered when deciding if a specific population is actually affected by cat predation. Cat diet was analysed on Santa Luzia (Cabo Verde Islands) since this invasive predator is considered one of the main threats to native endangered species that require conservation measures. These previous studies were carried out in different seasons, providing contrasting results, skinks being more preyed upon in the rainy season and mice in the driest periods. To check these different results, we focussed on how cat diet varied seasonally in response to changes in prey abundance. Saurians were the most important prey group, followed by mice, invertebrates and birds. No seasonal differences were, however, observed in the different prey groups consumed, saurians being the main prey in both seasons. All cases reflected their respective abundances. Results corroborate the generalist and opportunistic trophic ecology of feral cats, providing important information to assess their impact on prey populations and design future eradication programmes.     

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.