{"id":3501,"date":"2019-05-10T11:50:32","date_gmt":"2019-05-10T02:50:32","guid":{"rendered":"http:\/\/163.180.4.222\/lab\/?p=3501"},"modified":"2019-05-10T11:50:32","modified_gmt":"2019-05-10T02:50:32","slug":"remote-control-with-engineered-enzymes","status":"publish","type":"post","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=3501","title":{"rendered":"Remote control with engineered enzymes"},"content":{"rendered":"

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Many syntheses of organic molecules require that certain carbon-hydrogen bonds are targeted for reaction over others with similar reactivity (1<\/em><\/a>\u20136<\/em><\/a>). This high selectivity to one specific C\u2013H bond is frequently achieved by a remote activating group in the molecule (known as remote functionalization). A particularly attractive group of synthesis targets in pharmaceutical chemistry are chiral lactams, which are structural motifs in many bioactive compounds. The challenge is to design catalytic processes that can start from easily accessible derivatives of carboxylic acids and provide tunable control over stereoselective installation of the nitrogen at desired positions in the substrate, leading to lactams of varying ring size. On page 575 of this issue, Cho\u00a0et al.<\/em>\u00a0(7<\/em><\/a>) achieve this goal by harnessing the power of directed evolution. The authors engineered a toolbox of enzymes that are not found in nature for the remote C\u2013H functionalization of chemically diverse substrates to yield valuable lactam products.<\/p>\n

Cho\u00a0et al.<\/em>\u00a0used cytochrome P450 monooxygenases, a large class of enzymes involved in a wide range of oxidation reactions in nature. P450 monooxygenases contain a characteristic heme prosthetic group and can catalyze highly selective oxidations, such as the hydroxylation of nonactivated C\u2013H bonds, even when substrate flexibility makes selective functionalization more difficult. For example, Manning\u00a0et al.<\/em>\u00a0have recently reported the regio- and enantioselective midchain C\u2013H hydroxylation of simple fatty acid substrates by a native cytochrome P450 enzyme (8<\/em><\/a>).<\/p>\n

Hemeproteins, such as P450s, have also been the basis of new-to-nature enzymes, which catalyze reactions that have not been found in metabolic pathways. In earlier work, Arnold and coauthors took advantage of directed evolution technology to engineer cytochrome P450 variants, dubbed P411s, that catalyze the C\u2013H insertion of sulfonyl nitrenes to form C\u2013N bonds (9<\/em><\/a>). Singh\u00a0et al.<\/em>have shown that carbonyl-based nitrene donors can also be substrates for intramolecular C\u2013H amination processes (10<\/em><\/a>).<\/p>\n

Cho\u00a0et al.<\/em>\u00a0based their study on the recent discovery that a P450 is implicated in the biosynthesis of benzastatins through an acyl-protected hydroxylamine to form the reactive iron nitrenoid species (11<\/em><\/a>). The use of carbonyl-based nitrene donors opens the door to synthetically useful amides, but they are also susceptible to decomposition. Cho\u00a0et al.<\/em>\u00a0used directed evolution to develop P450-derived enzyme mutants that can direct carbonyl nitrenes to enantio- and regioselective C\u2013H amidation rather than the competing decomposition pathways.<\/p>\n

Cho\u00a0et al.<\/em>\u00a0selected \u03b2-lactams as the initial target products because of their prevalence in pharmaceutical applications. They identified a model substrate containing two sets of reactive C\u2013H bonds: one that could yield \u03b2-lactam as the C\u2013H amidation product, and another that could yield the corresponding \u03b4-lactam. They expressed a panel of hemeproteins, including native P450 enzymes and the P411 variants described in earlier work (9<\/em><\/a>), in\u00a0Escherichia coli<\/em>\u00a0cells. One P411 variant generated small amounts of \u03b2- and \u03b4-lactams and appeared to bypass any degradation to the corresponding amine by-product. The authors chose this variant as the parent enzyme for directed evolution experiments to create a more effective lactam synthase enzyme.<\/p>\n

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