Bioluminescence:Molecular Mechanisms and Biochemical Control

本研究将集中在海洋鞭毛藻（负责“海洋磷光”）和陆地双翅目（苍蝇）这两个主要的、进化上独立的生物发光生物群体的发光系统的基本生化机制上。发光反应的生化成分——荧光素酶（LCF）、荧光素底物（LH2）、独特的四吡咯和荧光素结合蛋白（LBP）——被隔离在一个细胞器（scintillon）中。LCF由三个高度保守的连续序列组成，每个序列都具有催化活性。活性所需的最小肽大小和活性位点的位置将被确定。与其他生物发光鞭毛藻的LCF序列进行比较将有助于鉴定酶活性和调控的保守残基，并将增加我们对荧光素酶基因结构进化的理解。长15毫米的北美食肉双翅目Platyura fultoni的幼虫有两个发光器官（头和尾），并发出蓝光，发光峰在460 nm处，但生物发光反应的生物化学机制尚不清楚。在所有的生物发光反应中，都需要氧气，这表明涉及过氧化物中间体。据报道，位于这些器官的巨细胞线粒体的颗粒分泌与发光有关。研究将集中在这些不寻常的细胞学方面和生物化学成分的鉴定。澳大利亚一种生物发光双翅目动物黄蜘蛛的织网幼虫，尽管在光器官解剖结构上存在差异，但与高原动物有相似之处；比较了这两种双翅目昆虫发光反应的生物化学性质。了解生物发光的基本机制将促进能量代谢的知识，以及我们对涉及活性氧的过程的理解，活性氧现在被认为是细胞信使和细胞损伤的媒介。对生物发光的基因、酶和底物的基础知识已经导致了广泛的应用，从基因表达的敏感可视化到药物和代谢物的分析测量。本文提出的鞭毛藻研究有望在酶学和分子进化方面取得重大进展。研究者最近开始研究的双翅目生物发光的生物化学是全新的，因此具有丰富的新知识和应用前景。该项目的具体目的如下：(a)研究其他生物发光鞭毛藻的荧光素酶基因，以深入了解基因的进化，并协助确定参与催化的氨基酸残基；(b)确定单个结构域内荧光素酶活性所需的结构区域；(c)表征双翅目板蓝目和蛛形目生物发光反应的生化成分，分离并部分纯化荧光素和荧光素酶；测试甲虫荧光素酶与双翅虫荧光素反应的能力。

This research will focus on the fundamental biochemical mechanisms of light emitting systems in marine dinoflagellates (responsible for the "phosphorescence of the sea") and terrestrial Diptera (flies), two major and evolutionarily independent groups of bioluminescent organisms. The biochemical components of the light-emitting reaction of the dinoflagellate Gonyaulax polyedra - the luciferase enzyme (LCF), the luciferin substrate (LH2), a unique tetrapyrrole, and a luciferin binding protein (LBP)- are sequestered in an organelle, the scintillon. LCF consists of three highly conserved and contiguous sequences, each of which is catalytically active. The minimum peptide size necessary for activity and the location of the active site will be determined. Comparison of LCF sequences from other bioluminescent dinoflagellates will allow residues conserved for enzyme activity and regulation to be identified, and our understanding of the evolution of the luciferase gene structure will be increased. Larvae of the web-building, carnivorous North American dipteran Platyura fultoni (15 mm long) have two light organs (head and tail) and emit a blue luminescence with an emission peak at 460 nm, but the biochemistry of the bioluminescence reaction is not known. Oxygen is required, suggesting the involvement of a peroxidic intermediate, as in all bioluminescence reactions. A granular secretion ascribed to the mitochondria of giant cells located in these organs is reported to be associated with the light emission. The study will focus on these unusual cytological aspects and on the identification of the biochemical components. The web-weaving larvae of an Australian bioluminescent dipteran, Arachnocampa flava, present similarities with Platyura despite differences in light organ anatomy; the biochemistry of the light emitting reactions of these two diptera will be compared. An understanding of the basic mechanisms in bioluminescence will advance the knowledge of energy metabolism, as well as our understanding of processes involving reactive oxygen species, now known to serve as cellular messengers as well as agents of cellular damage. Fundamental knowledge of the genes, enzymes and substrates responsible for bioluminescence has led to extensive applications, ranging from sensitive visualization of gene expression to analytical measurements of drugs and metabolites. The dinoflagellate studies proposed here promise to contribute significant advances in enzymology, as well as in molecular evolution. The biochemistry of the dipteran bioluminescence, which the investigator recently began studying, is entirely novel, hence rich in promises of new knowledge and applications. Specific aims for this project are as follows: (a) examine luciferase genes from other bioluminescent dinoflagellates so as to gain insight into gene evolution and to aid in the determination of amino acid residues involved in catalysis; (b) define the structural regions required for luciferase activity within the individual domains; (c) characterize the biochemical components of the bioluminescence reactions of the dipterans Platyura and Arachnocampa, isolate and partially purify luciferin and luciferase; test for the ability of the beetle luciferase to react with the dipteran luciferin.