Evolution of substrate specificity and chemical mechanism in thiamin diphosphate dependent enzymes

有了这个奖项，化学部的生命过程化学项目正在资助印第安纳州普渡大学印第安纳波利斯分校的Michael McLeish博士研究酶如何进化以选择其底物和产物。许多酶以二磷酸硫胺素的形式使用硫胺素作为辅助因子，以帮助它们加速生物反应。这些酶中有几种使用丙酮酸作为主要底物，但已经进化到将其转化为不同的产物。该项目将使用定向进化来产生使用丙酮酸的新硫胺素酶。稳态动力学和1H NMR和CD光谱的组合将用于确定这些新酶反应中各个化学步骤的速率常数，X射线晶体学将用于确定进化酶的结构。新酶的动力学将与其天然存在的对应物进行比较，并与这两种酶之间的任何结构差异相关。该项目的目标是对指导底物和反应特异性进化的原则有一个基本的理解。大多数酶都是非常专业的，并且已经进化到只识别一种或两种反应物并形成一种或两种产物。该项目将探索一组不寻常的酶能够以相同的方式使用相同的反应物进行操作，但将反应物转化为不同的产物。了解这个过程是重要的，因为这组酶可以用于工业上重要的反应。这项研究将需要改变底物特异性的酶的进化，并将使本科生和研究生有机会探索化学和生物过程。他们将接受微生物学、分子生物学、酶动力学、核磁共振光谱学和X射线晶体学方面的培训。重要的是，他们还将能够将这些研究与外展部分相结合，包括为中学生开发教学单元和动手酶学项目。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Michael McLeish from Indiana University-Purdue University Indianapolis to study how enzymes evolve to choose their substrate and products. Many enzymes use thiamin, in the form of thiamin diphosphate, as a cofactor to help them speed up biological reactions. Several of these enzymes use pyruvate as a primary substrate, but have evolved to convert it to different products. This project will use directed evolution to generate new thiamin enzymes that use pyruvate. A combination of steady-state kinetics and 1H NMR and CD spectroscopy will be used to determine rate constants for the individual chemical steps in the reactions of these new enzymes and X-ray crystallography will be used to determine the structures of the evolved enzymes. The kinetics of the new enzymes will be compared to those of their naturally-occurring counterparts and related to any structural differences between these two types of enzymes. The goal of the project is to attain a fundamental understanding of the principles guiding the evolution of substrate and reaction specificity.Most enzymes are very specialized and have evolved to recognize only one or two reactants and form one or two products. This project will explore the way in which an unusual group of enzymes is able to operate in the same manner using the same reactant, but turn the reactant into different products. Understanding this process is significant because this group of enzymes can be used in industrially important reactions. The study will require the evolution of enzymes with altered substrate specificity, and will allow undergraduate and graduate students an opportunity to explore both chemical and biological processes. They will receive training in microbiology, molecular biology, enzyme kinetics, NMR spectroscopy and X-ray crystallography. Importantly, they will also be able to integrate these studies with an outreach component that includes developing a teaching unit and a hands-on enzymology project for middle school students.