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

精细化工、制药和农化工业对生物催化剂的应用越来越感兴趣。2010年，全球工业酶市场总额为33亿美元，据预测，到2050年，30%的化工业务将是生物催化。开发更具选择性、更环保、更具成本效益的合成方法的需求推动了行业内的这种转变，开发新的酶势在必行。生活在冰冻环境中的细菌进化出分子适应能力，在低温环境中茁壮成长。它们通常表达在低温下有活性的酶，但在低于熔点的温度下，它们的中温和亲热对应物会展开。这些冷适应酶引起了生物技术行业的极大兴趣：它们在没有额外的热能输入的情况下实现了有用的催化速率，从而降低了制造成本；它们允许在增加其稳定性的条件下快速合成耐热性产品和中间体。尽管有这些优势，但我们对亲冷酶的物理化学性质和催化机理的了解仍落后于对中温酶和嗜热酶的了解。本项目将重点研究冷适应细菌酶atp -磷酸核糖基转移酶（ATPPRT）。该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-（5-磷酸核糖基）-ATP，并释放焦磷酸。该酶是一种杂聚低聚物，由催化亚基HisG和调节亚基HisZ组成，该亚基对组氨酸具有变构抑制作用。该项目的总体目标是揭示反应的催化机制，并利用这些知识来扩大其底物和产品范围。这将提供一种工具，使糖-核苷酸化学的快速多样化，同时揭示在低温下的酶催化。

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.