Enzyme studies for biocatalysis and other applications

生物催化和其他应用的酶研究

基本信息

  • 批准号:
    240750-2012
  • 负责人:
  • 金额:
    $ 5.1万
  • 依托单位:
  • 依托单位国家:
    加拿大
  • 项目类别:
    Discovery Grants Program - Individual
  • 财政年份:
    2015
  • 资助国家:
    加拿大
  • 起止时间:
    2015-01-01 至 2016-12-31
  • 项目状态:
    已结题

项目摘要

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.
酶引起我们研究小组的极大兴趣,不仅是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,在温和条件下,酶在广泛的反应中通常具有对映体、化学和区域选择性。然而,我们对这些上级催化剂的了解只是表面的。例如,在生物系统中发挥重要作用的变构性和协同性,只是定义得很差。我们在这里建议研究不同的酶,并利用它们的催化能力进行生物催化和其他应用。感兴趣的酶来自两组:1)一些乙酰基转移酶、磷酸基转移酶和腺苷酰转移酶,其显示新的变构行为;和2)P450酶,其显示复杂的协同行为并催化在失活的C-H键处的困难的氧化反应。 我们最近证明了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,Koshland-Nemethy-Filmer(KNF)-样机制反对Hilser-Hispsion(HT)-样机制,导致从积极到消极的合作行为,这取决于温度或配体。我们现在对这种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的相互作用。然而,需要对后者以及三元复合物(酶-乙酰辅酶A-氨基糖苷)进行更多的研究。使用动力学研究,以及ITC和NMR为基础的方法,最近在我们的小组,我们建议继续我们的研究这种酶,和其他酶怀疑表现类似(如一些氨基糖苷磷酸和腺苷转移酶)。 P450酶包括超过10,000个成员,其生物学作用范围从药物代谢到天然产物生物合成。它们因其与底物的同向和异向合作行为而闻名,并且还因其催化氧插入失活的C-H键的令人印象深刻的能力而闻名。存在非常少的允许脂肪族C-H键的直接羟基化的合成方法,并且它们中的大多数具有有限的范围。P450代表有前途的生物催化剂;然而,许多限制限制了它们的使用。这些包括底物特异性和产物可预测性,需要2或3个辅因子,与有机溶剂的不相容性和稳定性差。我们选择使用底物混杂的P450 3A 4,2D 6和2 E1,因为在研究环境中合成需要多功能,但可控的催化剂。我们已经报道了一些廉价的化学品可以有效地取代天然辅因子和条件,在有机溶剂的存在下使用这些酶。最近,我们已经开发了一个系统的底物标记,允许预测的区域和立体选择性的P450 3A 4。这些标签或化学助剂,一旦共价连接到底物,被设计为确保底物被酶识别,并定向其与酶的相互作用,以控制羟基化的区域和立体选择性。我们建议研究所观察到的选择性的机制,并扩大应用范围。例如,我们目前有2种酶/辅助系统,P450 3A 4/可可碱和P450 2 E1/烟酸,可以获得不同的产物(一种情况下,在辅助剂的第4个碳上进行Pro-R羟基化,另一种情况下,在倒数第二个碳上进行羟基化),但希望设计具有互补选择性的其他系统。 总的来说,这里提出的机械和工程研究的目的是提供一个更好的理解生物催化,和新的生物催化系统在研究中有用。

项目成果

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Auclair, Karine其他文献

Structure-Activity Relationships of Antiplasmodial Pantothenamide Analogues Reveal a New Way by Which Triazoles Mimic Amide Bonds
  • DOI:
    10.1002/cmdc.201800327
  • 发表时间:
    2018-12-20
  • 期刊:
  • 影响因子:
    3.4
  • 作者:
    Guan, Jinming;Tjhin, Erick T.;Auclair, Karine
  • 通讯作者:
    Auclair, Karine
Exploring Heteroaromatic Rings as a Replacement for the Labile Amide of Antiplasmodial Pantothenamides
  • DOI:
    10.1021/acs.jmedchem.0c01755
  • 发表时间:
    2021-04-01
  • 期刊:
  • 影响因子:
    7.3
  • 作者:
    Guan, Jinming;Spry, Christina;Auclair, Karine
  • 通讯作者:
    Auclair, Karine
Combining Small-Molecule Bioconjugation and Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to Expose Allostery: the Case of Human Cytochrome P450 3A4
  • DOI:
    10.1021/acschembio.1c00084
  • 发表时间:
    2021-04-29
  • 期刊:
  • 影响因子:
    4
  • 作者:
    Ducharme, Julie;Polic, Vanja;Auclair, Karine
  • 通讯作者:
    Auclair, Karine
Cellular Studies of an Aminoglycoside Potentiator Reveal a New Inhibitor of Aminoglycoside Resistance
  • DOI:
    10.1002/cbic.201800368
  • 发表时间:
    2018-10-04
  • 期刊:
  • 影响因子:
    3.2
  • 作者:
    Guan, Jinming;Vong, Kenward;Auclair, Karine
  • 通讯作者:
    Auclair, Karine
Small Molecule Restores Itaconate Sensitivity in Salmonella enterica: A Potential New Approach to Treating Bacterial Infections
  • DOI:
    10.1002/cbic.201600078
  • 发表时间:
    2016-08-17
  • 期刊:
  • 影响因子:
    3.2
  • 作者:
    Hammerer, Fabien;Chang, Justin H.;Auclair, Karine
  • 通讯作者:
    Auclair, Karine

Auclair, Karine的其他文献

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{{ truncateString('Auclair, Karine', 18)}}的其他基金

Rethinking biocatalysis and enzymology
重新思考生物催化和酶学
  • 批准号:
    RGPIN-2022-03032
  • 财政年份:
    2022
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual
Enzyme cooperativity, biocatalysis and bioconjugation
酶协同作用、生物催化和生物结合
  • 批准号:
    RGPIN-2017-04107
  • 财政年份:
    2021
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual
Enzymatic plastics depolymerization in moist-solid reaction mixtures
湿固体反应混合物中的酶促塑料解聚
  • 批准号:
    560533-2021
  • 财政年份:
    2021
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Idea to Innovation
Enzyme cooperativity, biocatalysis and bioconjugation
酶协同作用、生物催化和生物结合
  • 批准号:
    RGPIN-2017-04107
  • 财政年份:
    2020
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual
Enzyme cooperativity, biocatalysis and bioconjugation
酶协同作用、生物催化和生物结合
  • 批准号:
    RGPIN-2017-04107
  • 财政年份:
    2019
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual
Enzyme cooperativity, biocatalysis and bioconjugation
酶协同作用、生物催化和生物结合
  • 批准号:
    RGPIN-2017-04107
  • 财政年份:
    2018
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual
Enzyme cooperativity, biocatalysis and bioconjugation
酶协同作用、生物催化和生物结合
  • 批准号:
    RGPIN-2017-04107
  • 财政年份:
    2017
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual
Enzyme studies for biocatalysis and other applications
生物催化和其他应用的酶研究
  • 批准号:
    240750-2012
  • 财政年份:
    2016
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual
Enzyme studies for biocatalysis and other applications
生物催化和其他应用的酶研究
  • 批准号:
    240750-2012
  • 财政年份:
    2014
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual
Enzyme studies for biocatalysis and other applications
生物催化和其他应用的酶研究
  • 批准号:
    240750-2012
  • 财政年份:
    2013
  • 资助金额:
    $ 5.1万
  • 项目类别:
    Discovery Grants Program - Individual

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