Enzyme studies for biocatalysis and other applications

酶对我们小组非常感兴趣，不仅因为它们与疾病有关，而且还因为它们无与伦比的催化性能。事实上，在温和的条件下，酶在广泛的反应中往往是对映体、化学和区域选择性的。然而，我们对这些优秀催化剂的理解仅仅是表面上的。例如，变构和协作性在生物系统中扮演着重要角色，但对它们的定义并不明确。我们建议在这里研究不同的酶，并利用它们对生物催化和其他应用的催化能力。有兴趣的酶来自两类：1)一些表现出新的变构行为的乙酰基转移酶、磷酸基转移酶和腺基转移酶；2)P450酶，它们表现出复杂的合作行为，并在失活的C-H键上催化困难的氧化反应。 我们最近证实，屎肠球菌表达的氨基糖苷N-6‘-乙酰转移酶使用竞争性变构机制来调节底物结合。我们发现，类Koshland-Nemethy-Filmer(KNF)机制与类Hilser-Thompsion(HT)机制相反，导致合作行为从积极到消极不等，具体取决于温度或配体。我们现在对该酶与其底物乙酰辅酶A(AcCoA)之间的相互作用有了很好的了解，并开始了解它与另一种底物氨基糖苷类化合物的相互作用。然而，需要对后者以及三元复合体(酶-AcCoA-氨基糖苷)进行更多的研究。利用动力学研究以及我们团队最近开发的基于ITC和核磁共振的方法，我们建议继续对该酶以及其他被怀疑具有类似行为的酶(例如一些氨基糖苷磷酸化和腺苷转移酶)进行研究。 P450酶由10,000多个成员组成，其生物学作用范围从药物代谢到天然产物的生物合成。它们以其与底物的同向性和异向性合作行为而闻名，也因其令人印象深刻的催化氧插入失活的C-H键的能力而闻名。现有的允许脂肪族C-H键直接羟基化的合成方法很少，而且大多数方法的范围都很有限。P450代表着很有前途的生物催化剂；然而，一些限制限制了它们的使用。这些问题包括底物专一性和产品的可预测性、需要2到3个辅因子、与有机溶剂不相容以及稳定性差。我们选择使用底物混杂的P450 3A4、2D6和2E1，因为在研究环境中合成需要多功能但可控的催化剂。我们已经报道，在有机溶剂存在的情况下，一些廉价的化学品可以有效地取代天然辅因子和条件来使用这些酶。最近，我们开发了一种底物标记系统，可以预测P450 3A4的区域和立体选择性。这些标记或化学辅助剂一旦共价连接到底物上，就被设计来确保底物被酶识别，并定位它与酶的相互作用，以控制羟化的区域和立体选择性。我们建议研究观察到的选择性背后的机理，并扩大应用范围。例如，我们目前有两个酶/辅助系统，P450 3A4/可可碱和P450 2E1/烟酸，可以获得不同的产品(一种情况下是从辅助的4碳进行Pro-R羟基化，另一种是倒数第二碳的羟基化)，但希望设计出具有互补选择性的其他系统。 总体而言，这里提出的机械和工程研究旨在提供对生物催化的更好理解，以及在研究中有用的新的生物催化系统。

Enzymes are of great interest to our group, not only for their involvement in diseases but also for their unequalled catalytic properties. Indeed enzymes are often enantio-, chemo-, and regio-selective across a wide range of reactions under mild conditions. Yet our understanding of these superior catalysts is only superficial. For example, allostery and cooperativity, which play important roles in biological systems, are only poorly defined. We propose here to study different enzymes, and harness their catalytic abilities for biocatalyis and other applications. The enzymes of interest are from two groups: 1) some acetyltransferases, phosphoryltransferases and adenylyltransferases which show novel allosteric behavior; and 2) P450 enzymes which show complex cooperative behavior and catalyze difficult oxidation reactions at inactivated C-H bonds. We have recently demonstrated that the aminoglycoside N-6'-acetyltransferase expressed by Enterococcus faecium uses competing allosteric mechanisms to modulate substrate binding. We found that a Koshland-Nemethy-Filmer (KNF)-like mechanism opposes a Hilser-Thompsion (HT)-like mechanism, leading to cooperative behavior ranging from positive to negative, depending on the temperature or the ligand. We now have a very good understanding of the interaction between this enzyme and its substrate acetyl coenzyme A (AcCoA), and are starting to understand its interaction with aminoglycosides, the other substrate. More studies are however needed on the latter, as well as on the ternary complex (enzyme-AcCoA-aminoglycoside). Using kinetic studies as well as ITC- and NMR-based methods recently developed in our group, we propose to continue our studies of this enzyme, and of other enzymes suspected to behave similarly (e.g. some aminoglycoside phosphoryl and adenylyltransferases). P450 enzymes comprise more than 10,000 members with biological roles ranging from drug metabolism to natural product biosynthesis. They are well known for their homotropic and heterotropic cooperative behavior with substrates, and also for their impressive ability to catalyze the insertion of oxygen into inactivated C-H bonds. Very few synthetic methods exist that allow direct hydroxylation of aliphatic C-H bonds, and most of them are of limited scope. P450s represent promising biocatalysts; however, a number of limitations have restricted their use. These include substrate specificity and product predictability, the need for 2 or 3 cofactors, incompatibility with organic solvents, and poor stability. We elected to work with substrate-promiscuous P450s 3A4, 2D6 and 2E1 because synthesis in a research environment requires versatile, yet controllable catalysts. We have reported that some cheap chemicals can efficiently replace the natural cofactors and conditions to use these enzymes in the presence of organic solvents. More recently, we have developed a system of substrate tagging to allow prediction of the regio- and stereo-selectivity of P450 3A4. These tags or chemical auxiliaries, once covalently linked to the substrates, are designed to ensure that the substrate is recognized by the enzyme, and orient its interaction with the enzyme to control the regio- and stereo-selectivity of hydroxylation. We propose to study the mechanism underlying the observed selectivity and enlarge the scope of application. For example, we currently have 2 enzyme/auxiliary systems, P450 3A4/theobromine and P450 2E1/nicotinic acid, giving access to different products (Pro-R hydroxylation at the 4th carbon from the auxiliary in one case, and hydroxylation at the penultimate carbon in the other), but hope to design other systems with complementary selectivity. Overall the mechanistic and engineering studies proposed here aim at providing a better understanding of biocatalysis, and new biocatalytic systems useful in research.