Structure and function of the alcohol acyltransferases from yeast and fruit

挥发性酯是植物果实成熟过程中酵母在发酵过程中产生的重要次生代谢产物。因此，了解挥发性酯的生物化学在工业农业、酿酒和酿酒中具有相当重要的意义。此外，负责酯合成的酶现在正在细胞“工厂”中用于生产香料、工业溶剂、精细化学品和可再生生物燃料。然而，这些酶的详细结构和功能仍然知之甚少。这对在食品技术和合成生物学中开发这种酶提出了实质性的障碍。工业酿造酵母中有2个蛋白质家族负责挥发性酯的合成。这两个家族的功能都是酰基辅酶a：醇o -酰基转移酶，催化醇+酰基辅酶a共底物形成酯。我们最近从每个家族中确定了2个可以重组表达的模型蛋白，我们已经开始在体外研究它们。这个项目将建立在我们之前的工作的基础上，以表征这些酵母酶和相关的水果酰基转移酶的结构和功能。这是由3个相互关联的研究问题驱动的：酵母菌酰基转移酶对酰基辅酶a选择性的基础是什么？这两个酵母蛋白家族对不同链长的酰基辅酶a具有不同的活性谱。我们将研究这种特异性是如何在活性位点介导的。酵母酰基转移酶中硫酯酶活性的基础是什么？我们发现酵母酶对醇+是混杂的，出乎意料的是，在催化过程中，水可以代替醇；因此它们可以作为酰基辅酶a硫酯酶。我们将确定这一活动的机械基础。我们可以扩展我们的方法工具来研究水果酰基转移酶吗？这些酶具有重要的商业和生态意义，但尚未在体外进行有意义的表征。所有蛋白质将按照既定的方案进行重组表达和纯化，并使用最先进的机器人技术进入晶体屏幕。结合生化分析和气相色谱-质谱分析，研究酶活性和机制。计算配体对接将用于模拟和理解蛋白质与底物的相互作用。该项目将为学生提供高水平的技能培训：分子生物学；重组蛋白表达+纯化；生物化学+生物物理方法鉴定蛋白质；酶动力学;定点诱变;x射线结晶学;蛋白质建模+配体对接模拟。该项目直接关系到农业和粮食安全，特别是作物科学和健康安全食品领域。酒精酰基转移酶基因的表达与作物成熟直接相关，了解果实成熟的生物化学过程为通过选择性育种或基因改造改善和控制果实风味和品质提供了一条途径。因此，这与这些优先领域的几个目标有关：改变食物供应，延长保质期+提高消费者对水果的接受程度，以促进更健康的饮食。表征酵母的酰基转移酶也可能影响依赖酵母发酵的大众市场消费饮料的生产，如啤酒和葡萄酒。因此，这方面的影响可能包括降低成本+确保可用性。虽然与健康主题不太相关，但酵母衍生的发酵饮料是一个主要的工业问题；仅欧盟每年就生产近400亿升啤酒，销售额达1000亿欧元。当同样的技术可以应用于某些致病菌中必需的未表征的膜蛋白时，进一步的链被引入，因此对这种新型转运蛋白进行了首次生物物理研究，以阐明其结构和功能。

Volatile esters are important secondary metabolites that are produced by yeast during fermentation + in plants during fruit ripening.Understanding the biochemistry of the volatile esters is thus of considerable importance in industrial agriculture, winemaking + brewing.Additionally, the enzymes responsible for ester synthesis are now being used in cellular 'factories' to produce fragrances, industrial solvents, fine chemicals + renewable biofuels.However, the detailed structure and function of these enzymes remain poorly understood.This presents a substantial barrier to exploiting such enzymes in food technology and synthetic biology.There are 2 protein families in the industrial brewing yeast Saccharomyces cerevisiae responsible for volatile ester synthesis.Both families function as acyl-CoA: alcohol O-acyltransferases, catalysing ester formation from alcohol + acyl-CoA cosubstrates.We have recently identified 2 model proteins from each family that can be recombinantly expressed + we have begun to study them in vitro.This project will build upon our previous work to characterize the structure + function of these yeast enyzmes + of related acyltransferases from fruit.This is driven by 3 interrelated research questions:What is the basis for acyl-CoA selectivity by the yeast acyltransferases?The two yeast protein families have different activity profiles toward different chain-length acyl-CoAs.We will investigate how this specificity is mediated at the active site. What is the basis for thioesterase activity in the yeast acyltransferases?We have found that the yeast enzymes are promiscuous with regard to alcohol +,unexpectedly, that water can substitute for alcohol during catalysis; thus they can act as acyl-CoA thioesterases. We will determine the mechanistic basis of this activity. Can we extend our methodological toolkit to study fruit acyltransferases?These enzymes, with substantial commercial + ecological significance, have not yet been meaningfully characterised in vitro.All proteins will be recombinantly expressed + purified following established protocols and entered into crystal screens using state-of-the-art robotics.Coupled biochemical assays and GC-MS will be used to study enzyme activity + mechanism.Computational ligand docking will be used to simulate + understand protein-substrate interactions. This project will provide high-level skills training for the student:Molecular biology;recombinant protein expression + purification;protein characterization by biochemical + biophysical methods;enzyme kinetics;site-directed mutagenesis; X-ray crystallography; Protein modeling + ligand docking simulations. This project is directly relevant to Agriculture and Food Security, particularly the areas of Crop Science and Healthy + Safe Food.The expression of alcohol acyltransferase genes correlates directly with crop ripening, + understanding the biochemistry of fruit ripening offers a route towards improving + manipulating fruit flavour + quality via selective breeding or genetic modification.This is thus relevant to several of the goals of these priority areas: Changing food availability, increasing shelf life + increasing the consumer acceptance of fruits to promote a healthier diet. Characterising the acyltransferases from yeast could also influence the production of mass-market consumer beverages that rely upon yeast fermentation, such as beer + wine.Impacts in this arena are thus likely to include reducing cost + ensuring availability.Although less relevant to health themes, yeast-derived fermented beverages are a major industrial concern;the EU alone produces nearly 40bn litres of beer/annum with a sales value of >100bn euros. A further strand was introduced when evident the same techniques could be applied to an uncharacterized membrane protein that is essential in some pathogenic bacteria + thus pursued the first biophysical studies on this novel transport protein to elucidate structure + function.