Decoding of the cytochrome P450 monooxygenase CYP4B1 by biochemical, evolutional, and structural analysis

细胞色素P450家族4是最古老的P450酶家族之一;这些酶在整个进化过程中高度保守。在所有这些不同的酶中，本授权申请的主题，单加氧酶CYP 4 B1，是一种特殊和独特的酶，原因如下：到目前为止，还没有发现天然人CYP 4 B1酶的底物，因此将其定性为“孤儿”P450酶。人CYP 4 B1的催化活性明显缺失可部分归因于酶活性中心的所谓曲折区中的单个氨基酸变化：天然人CYP 4 B1在位置427处具有丝氨酸，而所有其他CYP 4 B1酶（以及哺乳动物中的所有CYP 4家族成员）在相应位置具有脯氨酸。在各种哺乳动物如牛、马、狗、大鼠、兔子或小鼠中，情况完全不同。在这里，CYP 4 B1作用于内源性和外源性代谢之间的界面：除了不饱和脂肪酸的ω-羟基化，CYP 4 B1还特别负责（与其他CYP 4家族成员相反）来生物活化一系列异生物质，包括4-异戊烯醇和紫苏酮。在进化过程中，在人类亚群、人类祖先和人类祖先中CYP 4 B1不同位置的遗传变化（Denisovan）和类人猿（黑猩猩）中大大减少或消除了CYP 4 B1的酶活性。基于HNO诊所和生物化学研究所之间的成功合作，该项目的主要目标是解码不同生物和物种进化过程中CYP 4 B1的生理重要性，并测试人类和大猿/灵长类动物中其他P450已经接管CYP 4 B1功能的假设。为了实现这一目标，将采取三种相互支持的方法：（i）将研究CYP 4 B1介导的代谢的功能方面，旨在找到CYP 4 B1的新底物。(ii)申请人将阐明CYP 4 B1的进化发展，并精确地确定CYP 4 B1功能丧失发生的时间线。此外，通过阐明其他细胞色素P450单加氧酶是否可以补偿丢失的CYP 4 B1功能，将研究对丢失的CYP 4 B1功能的进化补偿。(iii)最后，将研究CYP 4 B1与底物和抑制剂相互作用的结构方面，以补充和解释功能和进化研究的结果。这个串联项目的团队拥有所有必要的专业知识和合作者，可以深入分析这种孤儿CYP 4 B1的各个方面，并最终解码其生理功能。

The cytochrome P450 family 4 is one of the oldest P450 enzyme families; the enzymes have been highly conserved throughout evolution. Among all these different enzymes, the topic of this grant application, the monooxygenase CYP4B1, is a special and unique enzyme for the following reason: No substrate has been found for the native human CYP4B1 enzyme so far, thus qualifying it as an ‘orphan’ P450 enzyme. The apparent absence of catalytic activity of the human CYP4B1 can partially be attributed to a single amino acid change in the so-called meander region of the enzymes active centre: The native human CYP4B1 has a serine at position 427, whereas all other CYP4B1 enzymes (and also all CYP4 family members in mammals) have proline at the corresponding positions.Strikingly, the situation is completely different in various mammals such as cows, horses, dogs, rats, rabbits or mice. Here, CYP4B1 acts at the interface between endobiotic and xenobiotic metabolism: Besides ω-hydroxylation of unsaturated fatty acids, CYP4B1 is specifically responsible (in contrast to other CYP4 family members) to bioactivate of a range of xenobiotics including 4-ipomeanol and perilla ketone.However, previous data published by the applicants as well as unpublished preliminary data reveals a much more complex situation: During evolution, genetic changes at distinct positions in CYP4B1 in human subpopulations, human ancestors (Denisovan) and in great apes (chimps) greatly diminished or abolished the enzymatic activity of CYP4B1.Based on the successful collaboration between the HNO Clinic and the Institute of Biochemistry, the main objective of the project is to decode the physiological importance of CYP4B1 during evolution in different organisms and species, and to test the hypothesis that in humans and great apes/primates other P450s have taken over functions of CYP4B1. In order to reach this goal, three mutually supporting approaches will be undertaken: (i) Functional aspects of CYP4B1-mediated metabolism will be investigated aiming to find novel substrates for CYP4B1. (ii) The applicants will shed light on evolutionary development of CYP4B1 and precisely pinpoint the time-line at which losses of CYP4B1 functions occurred. Alongside, evolutionary compensation for lost CYP4B1 functions will be investigated by elucidating whether other cytochrome P450 monooxygenases can compensate for lost CYP4B1 functions. (iii) Finally, structural aspects of CYP4B1 interaction with substrates and inhibitors will be investigated to complement and explain the results of the functional and evolutionary investigations.The teams of this tandem project have all the necessary expertise and collaborators to profoundly analyse the described various aspects of this orphan CYP4B1 and finally decode its physiological function(s).