Mechanism and engineering of a cold-adapted hetero-oligomeric ATP-phosphoribosyltransferase: implications for synthetic biology

精细化学品、制药和农业化学工业对在工艺中利用生物催化剂越来越感兴趣。2010年，全球工业酶市场总额为33亿美元，预计到2050年，30%的化工业务将是生物催化。对开发更具选择性、更环保、更经济的合成方法的需求推动了这一行业的转变，开发新的酶势在必行。生活在冷冻环境中的细菌进化出了在低温下茁壮成长的分子适应性。它们通常表达在低温下有活性但在低于其嗜温和嗜热对应物的熔点的温度下展开的酶。这些冷适应酶吸引了生物技术行业的极大兴趣：它们在没有额外热能输入的情况下实现了有用的催化速率，这降低了制造成本;并且它们允许在增加其稳定性的条件下快速合成不耐热的产物和中间体。然而，我们对嗜冷酶的理化性质和催化机制的了解却远远落后于嗜温和嗜热酶。这种酶催化组氨酸生物合成的第一步，即5-三磷酸腺苷（ATP）和5-磷酸核糖基-1-焦磷酸（PRPP）缩合形成N1-组氨酸。（5-磷酸核糖基）-ATP，释放焦磷酸这种酶是由催化亚基HisG和调节亚基HisZ组成的异源寡聚体，该项目的首要目标是揭示该反应的催化机制，并利用这些知识来扩大其底物和产物范围。这将提供一种工具，使糖-核苷酸化学的快速多样化，同时阐明低温下的酶催化。

The fine chemicals, pharmaceutical, and agrochemical industries have an increasing interest in utilizing biocatalysts in processes. In 2010 the total global market for industrial enzymes was $ 3.3 billions, and it is predicted that by 2050, 30% of chemical business will be biocatalysis. The need to develop more selective, greener, and more cost-effective synthesis drives this shift within the industry, and it is imperative that new enzymes are developed.Bacteria that dwell in frozen environments evolved molecular adaptations to thrive in low temperature. They often express enzymes that are active at low temperature but unfold at temperatures below the melting point for their mesophilic and thermophilic counterparts. These cold-adapted enzymes attract significant interest from the biotechnology industry: they achieve useful rates of catalysis without additional thermal energy input, which reduces manufacturing costs; and they allow thermolabile products and intermediates to be rapidly synthesized under conditions that increases their stability. In spite of these advantages, our knowledge about the physical chemical properties and catalytic mechanisms of psychrophilic enzymes lags behind that about mesophilic and thermophilic ones.This project will focus on the cold-adapted bacterial enzyme ATP-phosphoribosyltransferase (ATPPRT). This enzyme catalyses the first step of histidine biosynthesis, the condensation of adenosine 5-triphosphate (ATP) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to form N1-(5-phosphoribosyl)-ATP, with release of pyrophosphate This enzyme is a hetero-oligomer consisting of a catalytic subunit, HisG, and a regulatory subunit, HisZ, which confers allosteric inhibition by histidine.The overarching aim of the project is to unravel the catalytic mechanism of the reaction and harness that knowledge to expand its substrate and product scope. This will provide a tool to enable rapid diversification of sugar-nucleotide chemistry while shedding light on enzymatic catalysis at low temperature.