CAREER: Mechanistic and Biosynthetic Studies of Dinoflagellate Bioluminescence

通过这一奖项，化学部的生命过程化学项目资助了奥本大学的Steven O. Mansoorabadi博士，他的研究项目专注于鞭毛藻生物发光的机理和生物合成研究。鞭毛藻是淡水和海洋环境中发现的重要微生物群。某些鞭毛藻产生强效毒素并引起赤潮，对沿海生态系统以及人类和海洋野生动物的健康产生重大负面影响。有几种鞭毛藻既能进行光合作用又能发光，它们是海洋发出明亮蓝色光芒的原因。鞭毛藻利用一种被称为鞭毛藻荧光素酶的酶对物理搅动产生光。这种酶的底物荧光素是由叶绿素通过一种未知的途径产生的。本课题旨在通过阐明甲藻荧光素酶的发光机制和荧光素的生物合成途径，更好地了解甲藻生物发光系统。这些研究有助于促进鞭毛藻荧光素酶作为细胞显像剂的使用，并可能导致用于沿海海水修复的杀藻剂的发展。该项目还将生物发光这一令人兴奋和迷人的自然现象整合到两个K-12外展计划中，旨在吸引和激励年轻学生从事科学和相关领域的职业。它还将包括奥本大学暑期科学研究所的学生，该研究所针对有天赋的高中生和非盟探索计划，该计划主要面向农村学校的中学生。鞭毛藻荧光素酶是研究ph依赖性酶调节和生物化学发光的化学机制的模型系统，这两者都不是很清楚。为了深入了解鞭毛藻荧光素酶的调控，使用时间分辨光热方法、恒定pH分子动力学模拟和氢/氘交换质谱法确定了pH诱导的构象变化的幅度和时间尺度以及相关的结构和动力学变化。通过结合瞬态动力学和光谱方法以及随时间变化的密度泛函理论计算，确定发光团和其他关键催化中间体的结构，研究了鞭毛藻荧光素酶产生光的详细机制。此外，使用突变分析确定了对底物结合和催化重要的活性位点残基的身份。最后，通过鉴定的生物合成中间体和酶的生化和结构表征，深入了解叶绿素分解代谢导致鞭毛藻荧光素形成的途径。总之，这些研究为这些重要的海洋微生物的生物化学提供了重要的见解，并为本科生和研究生提供了极好的跨学科培训机会。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Steven O. Mansoorabadi from Auburn University, whose research project is focused on mechanistic and biosynthetic studies of dinoflagellate bioluminescence. Dinoflagellates are an important group of microorganisms found in freshwater and marine environments. Certain dinoflagellates produce potent toxins and cause red tides, which have a significant negative impact on coastal ecosystems and the health of humans and marine wildlife. Several species of dinoflagellates are both photosynthetic and bioluminescent, and are responsible for the bright blue glow of the sea. Dinoflagellates produce light in response to physical agitation using an enzyme known as dinoflagellate luciferase. The substrate of this enzyme, luciferin, is produced from chlorophyll by an unknown pathway. This research project aims to provide a better understanding of the dinoflagellate bioluminescence system by elucidating the mechanism of light production by dinoflagellate luciferase and the biosynthetic pathway for the production of luciferin. These studies help facilitate the use of dinoflagellate luciferase as a cellular imaging agent, and may lead to the development of algicides for the remediation of coastal seawater. This project also integrates bioluminescence, an exciting and fascinating natural phenomenon, into two K-12 outreach initiatives designed to attract and inspire young students to pursue careers in science and related fields. It will also include students in the Auburn University Summer Science Institute directed at gifted high school students and the AU Explore a program that engages middle school students from predominantly rural schoolsDinoflagellate luciferase is a model system for the study of both pH-dependent enzyme regulation and the chemical mechanism of biological chemiluminescence, neither of which are well understood. To gain insight into the regulation of dinoflagellate luciferase, the magnitude and timescale of the pH-induced conformational change and the associated variations in structure and dynamics is determined using time-resolved photothermal methods, constant pH molecular dynamics simulations, and hydrogen/deuterium exchange mass spectrometry. The detailed mechanism by which dinoflagellate luciferase produces light is investigated by determining the structure of the luminophore and other key catalytic intermediates using a combination of transient kinetic and spectroscopic methods and time-dependent density functional theory calculations. In addition, the identity of active site residues important for substrate binding and catalysis is ascertained using mutational analysis. Finally, insight into the pathway of chlorophyll catabolism leading to the formation of dinoflagellate luciferin is obtained through the biochemical and structural characterization of identified biosynthetic intermediates and enzymes. Together, these studies provide significant insight into the biochemistry of these important marine microorganisms and afford excellent interdisciplinary training opportunities for undergraduate and graduate students.