Evaluating Aspects of O2-Activation by Bacterial Multicomponent Monooxygenases

说明(由申请人提供)：随着市政供水中卤代有机物(据称致癌物质)水平的增加，对公众健康的风险也增加；因此，需要开发方法来降解这些材料。细菌多组分单加氧酶(BMMS)是一种典型的由羟基酶、还原酶和调节蛋白组成的酶，近年来被研究作为生物修复试剂。因此，了解BMM催化的各个方面对于开发降解污染物的酶至关重要。尽管甲烷单加氧酶已经被研究了几十年，但质子和电子传递到BMMS活性中心的机制仍然是一个谜。借助苯酚羟基酶(PHH)及其调节蛋白(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，调节蛋白以及潜在的还原酶覆盖着一个广泛的氢键网络(在BMMS中保守)，该网络从PHH表面开始并延伸到活性部位。氢键网络中涉及的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活性部位的结构完整性中的作用。我们将监测突变对PH的催化活性和稳定性的影响。罗氏酵母B-276的烯烃单加氧酶(AMO)是一种能够环氧化烯烃的BMM，它将被克隆到为表达多亚单位酶而开发的大肠杆菌载体中。这将有助于首次对AMO进行结构研究。典型的BMM羟基酶有a2？2？2异二聚体组成，而AMO只有a和？亚基。由于β-亚基在BMMS中的位置和折叠有很大的变异性，而且AMO与BMM相比具有更高的立体选择性和区域选择性，我们认为β-亚基可能起着重要的作用。与公众健康相关由于细菌单加氧酶能够降解广泛的废水污染物，因此改造这些酶以更好地适应这一角色的潜力是非常有意义的。通过这项工作了解这些酶的O2-激活和底物专一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.