Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
J Am Chem Soc. 2010 Oct 27;132(42):14697-9. doi: 10.1021/ja107344h.
The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ∼3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS β-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.
模块 4 的脱水酶 (DH) 结构域已被证明可催化 6-脱氧赤藓醇 B 合酶 (DEBS) 中独特的顺式消除/顺式加成水反应。将重组 DH4 与通过化学酶法制备的反式-(2R,3R)-2-甲基-3-羟基戊酰-ACP（2a-ACP）孵育，得到脱水产物 3-ACP。同样，DH4 与合成的 3-ACP 孵育导致相反的酶催化水合反应，得到 2a-ACP 和 3-ACP 的约 3:1 平衡混合物。将丙酰基-SNAC（4）、甲基丙二酰辅酶 A 和 NADPH 与 DEBS β-酮酰基合酶-酰基转移酶[KS6][AT6]双结构域、DEBS ACP6 和泰乐菌素合酶模块 1 的酮还原酶结构域（TYLS KR1）一起孵育，原位生成反式-2a-ACP，该反式-2a-ACP 经 DH4 催化顺式脱水生成 3-ACP。DH4 不能使 DEBS KR1、DEBS KR6 或利福霉素合酶 KR7（RIFS KR7）原位生成的顺式-(2S,3R)-2b-ACP、顺式-(2R,3S)-2c-ACP 或反式-(2S,3S)-2d-ACP 发生脱水反应。同样，将（2S,3R）-2-甲基-3-羟基戊酰基-N-乙酰半胱氨酸硫酯（2b-SNAC）、甲基丙二酰辅酶 A 和 NADPH 与 DEBS [KS6][AT6]、DEBS ACP6 和 TYLS KR1 一起孵育，得到反式-(2R,3R)-6-ACP，该反式-(2R,3R)-6-ACP 经 DEBS DH4 催化顺式脱水生成（4R,5R）-(E)-2,4-二甲基-5-羟基-庚-2-烯酰基-ACP（7-ACP）。通过 GC-MS 和 LC-MS 对衍生的甲酯 7-Me 与合成的 7-Me 样品进行比较，确定了 7 的结构和立体化学。
