CATALYTIC DOMAIN OF CHICKEN TRYPTOPHAN HYDROXYLASE ISOFORM 1

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 抑郁症、进食障碍、精神分裂症、强迫症和许多其他神经精神疾病可能是由于大脑中神经递质5-羟色胺水平失衡引起的。含铁酶色氨酸羟基酶(TPH)催化5-羟色胺生物合成的限速步骤，因此了解该酶的结构和功能对于从分子上理解上述疾病是重要的。TPH的完整机制尚不清楚。众所周知，TPH不稳定且难以纯化，目前只发表了这种双截断酶的5种晶体结构。 我们团队已经开发了几种方法来纯化几种TPH变体的稳定溶液，这些变体现在已经结晶。一种结构已经被解决，并于2008年发表在《生物化学》(PDB ID 3E2T)上。这是芳香族氨基酸羟基酶的第一个与天然底物结合的结构。现在我们有了TPH的相同变体的晶体，只有底物(以前的结构也包含来自结晶溶液的非自然配体)，并与底物和辅因子结合。这些结构的确定将使我们能够研究底物和辅因子结合时发生的结构变化。结合斯坦福大学所罗门实验室收集的CD/MCD数据，这些结构将给出TPH1中结构/功能关系的完整图景。TPH1的晶体在40到100微米之间，清楚地是三维的，形状很好，边缘光滑。最后的晶体衍射率达到1.9°，然而这些新晶体出现的时间较短(3天比6个月)，因此实际分辨率尚不清楚。由于我们为两种可能的结构各有几个晶体，因此使用自动安装机来寻找最佳晶体的过程将是更可取的。这些结构将通过使用先前结构(3E2T)的分子置换来解决。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Depression, eating disorders, schizophrenia, OCD (obsessive compulsive disorder) and many other neuropsychiatric disorders are presumably caused by imbalance in the level of the neurotransmitter serotonin in the brain.The iron-containing enzyme tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin and knowledge on the structure and function of this enzyme is thus important for the molecular understanding of the disorders mentioned above. The complete mechanism of TPH is not known.TPH is known to be notoriously unstable and difficult to purify, and only 5 crystal structures of the double truncated enzyme have been published. Our group has developed methods to purify stable solutions of several TPH variants, which have now been crystallized. One structure has already been solved and was published in Biochemistry in 2008 (PDB ID 3E2T). This was the first structure of any aromatic amino acid hydroxylase with the natural substrate bound. Now we have crystals of this same variant of TPH, with only substrate (the previous structure also contained an unnatural ligand from the crystallization solution) and with substrate and cofactor bound. Determination of these structures will allow us to investigate the structural changes which occur upon substrate and cofactor binding. Together with CD/MCD data collected at The Solomon Laboratory at Stanford University, the structures will give a complete picture of the structure/function relationship in TPH1. The crystals of TPH1 are between 40 and 100 um, clearly three dimensional and nicely shaped with smooth edges. The last crystal diffracted to a resolution of 1.90 ¿, however these new crystals appeared after shorter time (3 days vs. 6 months) so the actual resolution is not known.As we have several crystals for each of the two possible structures, a scouting process for the-best-in-the-lot using automounters would be preferable. The structures will be solved with molecular replacement using the previous structure (3E2T).