An acetylation/deacetylation cycle controls the export of sterols and steroids from S. cerevisiae

Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. Here we report the identification of a novel reversible sterol modification in yeast, the sterol acetylation/deacetylation cycle. Sterol acetylation requires the acetyltransferase ATF2, whereas deacetylation requires SAY1, a membrane-anchored deacetylase with a putative active site in the ER lumen. Lack of SAY1 results in the secretion of acetylated sterols into the culture medium, indicating that the substrate specificity of SAY1 determines whether acetylated sterols are secreted from the cells or whether they are deacetylated and retained. Consistent with this proposition, we find that acetylation and export of the steroid hormone precursor pregnenolone depends on its acetylation by ATF2, but is independent of SAY1-mediated deacetylation. Cells lacking Say1 or Atf2 are sensitive against the plant-derived allylbenzene eugenol and both Say1 and Atf2 affect pregnenolone toxicity, indicating that lipid acetylation acts as a detoxification pathway. The fact that homologues of SAY1 are present in the mammalian genome and functionally substitute for SAY1 in yeast indicates that part of this pathway has been evolutionarily conserved.     

Specific recognition of saturated and 4,5-unsaturated hexuronate sugars by a periplasmic binding protein involved in pectin catabolism

The process of pectin depolymerization by pectate lyases and glycoside hydrolases produced by pectinolytic organisms, particularly the phytopathogens from the genus Erwinia, is reasonably well understood. Indeed each extracellular and intracellular catabolic stage has been identified using either genetic, bioinformatic or biochemical approaches. Nevertheless, the molecular details of many of these stages remain unknown. In particular, the mechanism and ligand binding profiles for the transport of pectin degradation products between cellular compartments remain entirely uninvestigated. Here we present the structure of TogB, a 45.7 kDa periplasmic binding protein from Yersinia enterocolitica. This protein is a component of the TogMNAB ABC transporter involved in the periplasmic transport of oligogalacturonides. In addition to the unliganded complex (at 2.2 A), we have also determined the structures of TogB in complex with digalacturonic acid (at 2.2 A), trigalacturonic acid (at 1.8 A) and 4,5-unsaturated digalacutronic acid (at 2.3 A). The molecular determinants of oligogalacturonide binding include a novel salt-bridge between the non-reducing sugar uronate group, selectivity for the unsaturated ligand, and the overall sugar configuration. Complementing this are UV difference and isothermal titration calorimetry experiments that highlight the thermodynamic basis of ligand specificity. The ligand binding profiles of the TogMNAB transporter complex nicely complement pectate lyase-mediated pectin degradation, which is a significant component of pectin depolymerization reactions.     

A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7

We have characterized an esterase expressed from the putative esterase gene (ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli. The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration. The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 degrees C and pH 7.5-8.0. The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide. From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.     

摩氏摩根菌产酯酶条件优化及在普瑞巴林手性中间体合成中的应用

为建立廉价、高效的普瑞巴林关键手性中间体生产工艺,经单因素优化和正交试验,确定了摩氏摩根菌CCTCC M 2011175最佳产酯酶培养基,组成(g/L)为葡萄糖15.0,牛肉膏7.0,Na2HPO41.0,Fe2(SO4)30.1,吐温-8010.0。优化后酯酶比酶活达到1 071.0 U/L,为出发培养基的2.5倍。以培养基优化后获得的摩氏摩根菌菌体为催化剂,立体选择性拆分外消旋2-羧乙基-3-氰基-5-甲基己酸乙酯水解,转化率达到45%,对映体过量值(e.e.)大于94%,为酶法生产普瑞巴林关键手性中间体奠定了良好基础。     

The feruloyl esterase gene family of Fusarium graminearum is differentially regulated by aromatic compounds and hosts

Feruloyl esterases can liberate ferulic acid (FA) from plant cell wall polymers. They are expressed by plant pathogenic fungi and could play a role in pathogenicity, although this question has not been addressed yet. The fungus Fusarium graminearum is the principal causal agent of fusarium head blight (FHB) and gibberella ear rot (GER), major diseases of wheat, barley, and maize in all temperate regions of the world. The F. graminearum genome contains seven genes with strong homology to feruloyl esterase (FAE) sequences. Phylogenetic analysis showed that these included three type B, three type C, and one type D FAE genes. Expression profiling of the seven FAE genes showed complex regulation patterns unique to each gene. In F. graminearum-infected plant tissues, the FAE genes exhibited host-specific gene expression. On wheat, FAEB1 and FAED1 were strongly expressed while FAEB2, FAEB3, and FAEC1 were expressed at more modest levels. On maize, only FAEB3, FAEC1, and FAED1 were expressed and at low levels. When growing F. graminearum in liquid culture, only FAEB1 and FAEC1 were expressed. Both genes were induced by a small group of related aromatic compounds including FA, caffeic acid, and p-coumaric acid. FAEB1 was induced by xylose, while repressed by glucose and galactose. FAEC1 was constitutively expressed at low levels in the presence of those sugars. Expression of the other five FAE genes was not detected in the culture conditions used. To determine if FAE genes were important for pathogenicity of F. graminearum, mutant strains inactivated for faeB1?, faeD1? or both genes were constructed and tested on wheat plants. No statistically significant change in pathogenicity and no compensatory expression of the other FAE genes were observed in the fae gene mutants. Our results show that FAEB1 and FAED1 are not required for pathogenicity of F. graminearum on wheat.     

培养时间对黑木耳酯酶同工酶谱的影响

为准确地将酯酶同工酶技术应用于食用菌菌株鉴别和遗传育种研究以及进行快速的菌种区分、鉴定,本试验对10个黑木耳菌株不同培养时期的胞内酯酶(EST)同工酶谱进行了研究,并对菌株间亲和性试验结果进行分析。结果表明:同一培养时期的各菌株间同工酶谱存在明显差异,不同培养时期同一菌株同工酶谱也存在一定的差异。培养20d诱导的同工酶谱可有效区分、鉴定各菌株。聚类分析结果表明:培养20d的酶谱聚类分析结果与亲和性试验结果相一致。     

农药残留检测用植物酯酶的筛选

基于酶法分析中的酶抑制原理,通过对8种植物种子及2种植物加工材料总酯酶活力及其比活力进行比较,筛选农药残留检测用适宜的植物酯酶。结果显示,花豇豆总酯酶活力和比活力分别达7.325 U和0.617 U/mg,显著高于其它9个实验材料。用花豇豆酯酶对6种有机磷及氨基甲酸酯类农药——敌敌畏、辛硫磷、敌百虫、速灭威、克百威、灭多威进行敏感性分析,最低检测限均比国家规定的最大残留量小,符合检测要求。结果表明,花豇豆的酯酶总活力、比活力及其敏感性最好,是一种农药残留检测的理想酶源植物。