Evaluating Aspects of O2-Activation by Bacterial Multicomponent Monooxygenases

描述（由申请人提供）：随着在市政供水中发现的卤化有机物（所谓的致癌物）含量的增加，对公众健康的风险也在增加；因此，开发降解这些材料的方法是至关重要的。细菌多组分单加氧酶（BMMs）是一种典型的由羟化酶、还原酶和调节蛋白组成的酶，近年来被研究作为生物修复剂。因此，了解BMM催化的各个方面对于开发降解污染物的酶是必不可少的。虽然对甲烷单加氧酶的研究已经有几十年的历史了，但其向bmp活性位点传递质子和电子的机制仍然是一个谜。借助最近苯酚羟化酶（PHH）及其调控蛋白（PHM）的晶体结构，我们提出了几种突变来解决这些问题。当与PHH结合时，调节蛋白和潜在的还原酶覆盖了一个广泛的氢键网络（在bmm中保守），该网络从PHH表面开始延伸到活性位点。氢键网络中残基的突变将使我们能够确定该网络在质子转移、电子转移和PHH活性位点结构完整性中的作用。我们将监测突变对ph催化活性和稳定性的影响。从R. Rhodochrous B-276（一种能够环氧化烯烃的BMM）中克隆出的烯单加氧酶（AMO）将被克隆到一个用于表达多亚基酶的大肠杆菌载体中。这将有助于对AMO进行首次结构研究。通常BMM羟化酶具有a2¿2？2异二聚体组成，但AMO只有a和¿亚基。因为在位置和折叠上有太多的可变性？与BMM相比，AMO具有较高的立体选择性和区域选择性，我们认为？-亚基可能起着重要作用。由于细菌单加氧酶能够降解多种废水污染物，因此设计这些酶以更好地适应这一作用的潜力引起了极大的兴趣。从这项工作中了解这些酶的o2活化和底物特异性的机制将促进基础知识的发展，并进一步发展这些酶作为废水处理目的的生物催化剂。

DESCRIPTION (provided by applicant): As the levels of halogenated organics (alleged carcinogens) found in municipal water supplies increase, the risk to public health also increases; therefore, the need to develop ways to degrade these materials is of utmost importance. Bacterial multicomponent monooxygenases (BMMs), enzymes typically consisting of a hydroxylase, a reductase and a regulatory protein, have recently been studied as bioremediation agents. Understanding aspects of BMM catalysis is thus essential for the development of enzymes for the degradation of pollutants. Although methane monooxygenase has now been studied for several decades, the mechanism of proton and electron delivery to the active sites of BMMs is still somewhat of a mystery. With the help of the recent crystal structure of phenol hydroxylase (PHH) and its regulatory protein (PHM) we have proposed several mutations to address these issues. When bound to PHH the regulatory protein, and potentially the reductase, covers an extensive hydrogen-bonding network (conserved in BMMs) that starts at the surface of PHH and extends into the active site. Mutation of residues involved in the hydrogen-bonding network will enable us to determine the role of this network in proton transfer, electron transfer, and structural integrity of the PHH active site. We will monitor the effects of the mutations on the catalytic activity and stability of PH. Alkene monooxygenase (AMO) from R. Rhodochrous B-276, a BMM capable of epoxidation of alkenes, will be cloned into an E. coli vector developed for the expression of multisubunit enzymes. This will facilitate the first structural studies on AMO. Typically the BMM hydroxylases have an a2¿2?2 heterodimeric composition, but AMO only has an a and ¿ subunit. Because there is so much variability in the location and fold of the ?-subunits in BMMs, and AMO performs reactions with high stereo- and regioselectivity in comparison to its BMM counterparts, we believe that the ?-subunit may be playing an important role. PUBLIC HEALTH RELEVANCE Because bacterial monooxygenases are capable of the degradation of a wide range of wastewater pollutants, the potential to engineer these enzymes to be better suited for this role is of great interest. Understanding the mechanisms of O2-activation and substrate specificity in these enzymes from this work will both advance fundamental knowledge and further the development of these enzymes as biocatalysts for wastewater treatment purposes.