Outer sphere contributions within the thiol dioxygenase active site: a combined structure-function study

标题.硫醇双加氧酶活性位点的外球贡献：结构-功能组合 研究 项目摘要。巯基双加氧酶（TDO）是非血红素单核铁加氧酶的一个亚类 其催化含硫醇底物的O2依赖性氧化以产生相应的亚磺酸。 半胱氨酸双加氧酶（CDO）和半胱胺双加氧酶（ADO）是已知的仅有的哺乳动物TDO。这些 酶催化L-半胱氨酸（CYS）和半胱胺（2-氨基乙硫醇，CA）氧化产生半胱氨酸 亚磺酸（CSA）和亚牛磺酸（HT）。最终，CSA和HT进入哺乳动物体内， 牛磺酸（TAU）的生物合成途径。作为体内最丰富的（磺酸）氨基酸衍生物， TAU具有多种重要功能，包括转录调控、结合底物等，在合成 胆盐，稳定骨骼肌，维持心律，以及一种重要的神经递质， 胚胎中枢神经系统发育尽管CDO和ADO具有结构相似的底物， 表现出显著的底物特异性;表现出无（或可忽略的）交叉反应性。值得注意的是， 哺乳动物TDO（CDO和ADO）的细胞表达和/或活性与 各种人类疾病（癌症、神经退行性疾病、类风湿性关节炎以及其他代谢性疾病）的治疗。 疾病）。TDO的新兴子集被称为N-末端半胱氨酰双加氧酶（NCO）。这些 酶被认为通过催化蛋白质N-末端的氧化而起到生理O2-传感器的作用 启动蛋白质降解的CYP-残基。最近报道了哺乳动物ADO的类似功能 控制G蛋白信号的调节器。因此，ADO既作为小分子TDO，又作为特异性TDO， 对于半胱胺（CA），以及NCO，通过以O2依赖性方式调节特定蛋白质的稳定性。 NCO活性位点内的一个显著区别是不存在空间相邻的保守序列。 形成氢键网络的氨基酸（Ser-His-Tyr）。这种结构基序在以下物种中高度保守： 结构上表征的TDO，并且据信其调节氧和硫醇-底物结合亲和力， TDO Fe现场。这种保守序列在NCOs中的缺失表明了一种新的O2传感机制 在这类酶中。拟议活动的中心主题是调查外层空间的作用 TDO活性位点内的相互作用影响底物效率和识别。实验 建议涉及一系列仔细的动力学测量，以确定催化内的基本残留物， 酶活性位点同时，光谱和计算方法将用于开发一个 活性部位氢键相互作用的结构模型。类似的研究将在以下成员身上进行： NCO类酶提供了一个有价值的对应点，为TDO获得的结果。潜在这些 研究将阐明TDO（和NCO）调节小分子特异性的机制， 硫醇、多肽底物和分子氧。

TITLE. Outer sphere contributions within the thiol dioxygenase active site: a combined structure-function study PROJECT SUMMARY. Thiol dioxygenases (TDOs) are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. Cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO) are the only known mammalian TDOs. These enzymes catalyze the oxidation of L-cysteine (CYS) and cysteamine (2-aminoethanthiol, CA) to produce cysteine sulfinic acid (CSA) and hypotaurine (HT), respectively. Ultimately, CSA and HT feed into the mammalian biosynthetic pathways for taurine (TAU). As the most abundant (sulfonic) amino acid derivative in the body, TAU plays a variety of essential functions including osmoregulation, conjugation substrate in the synthesis of bile salts, stabilization of skeletal muscle, maintenance of cardiac rhythm, and an essential neurotransmitter in embryonic central nervous system development. Despite having structurally similar substrates, CDO and ADO exhibit remarkable substrate-specificity; exhibiting no (or negligible) cross-reactivity. Significantly, changes in the cellular expression and/or activity of mammalian TDOs (CDO and ADO) have been correlated with the onset of various human diseases (cancer, neurodegenerative disorders, rheumatoid arthritis, as well as other metabolic disorders). An emerging subset of TDOs are referred to as N-terminal cysteinyl dioxygenases (NCOs). These enzymes are believed to function as physiologic O2-sensors by catalyzing the oxidation of protein N-terminal CYS-residues to initiate protein degradation. A similar function has recently been reported for mammalian ADO in controlling regulators of G protein signaling. Therefore ADO functions both as a small molecule TDO, specific for cysteamine (CA), as well as a NCO by regulating the stability of specific proteins in an O2-dependent fashion. A notable distinction within the NCO active site is the absence of a conserved sequence of spatially adjacent amino acids (Ser-His-Tyr) forming a hydrogen bonding network. This structural motif is highly conserved among structurally characterized TDOs and it is believed to regulate oxygen and thiol-substrate binding affinity at the TDO Fe-site. The absence of this conserved sequence among NCOs suggests a novel mechanism for O2-sensing in this class of enzymes. The central theme of the proposed activities is to investigate the role of outer-sphere interactions within the TDO active site that influence substrate efficiency and recognition. The experiments proposed involve a series of careful kinetic measurements to identify catalytically essential residues within the enzymatic active site. In parallel, spectroscopic and computational methods will be employed to develop a structural model for active site hydrogen-bonding interactions. Similar studies will be performed on members of the NCO class of enzymes to provide a valuable counterpoint to results obtained for TDOs. Potentially these studies will shed light on the mechanisms by which TDOs (and NCOs) regulate specificity of small molecule thiols, polypeptide substrates, and molecular oxygen.