Probing the acetaldehyde-sensitivity of 2-deoxy-ribose-5-phosphate aldolase (DERA) leads to resistant variants
Probing the acetaldehyde-sensitivity of 2-deoxy-ribose-5-phosphate aldolase (DERA) leads to resistant variants
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DOI：
10.1016/j.jbiotec.2017.03.024
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发表时间：
2017-09-20
影响因子：
4.1
通讯作者：
Classen, Thomas
中科院分区：
文献类型：
--
作者：
Bramski, Julia;Dick, Markus;Classen, Thomas
2-脱氧-O-核糖-5-磷酸醛缩酶（DERA）是一种合成上有吸引力的酶，因为它能够立体选择性地进行C-C-偶联，该酶使用乙醛作为亲核试剂，因此产生真正的醛醇而不是酮醇，并且可以将两个乙醛分子添加到一个亲电试剂上。然而，DERA产生巴豆醛作为乙醛的副反应，乙醛然后是在活性位点内形成共价迈克尔加合物的不可逆抑制剂，特别是与半胱氨酸47（Dick等人，2016）。这种抑制可以通过将C47突变为非亲核氨基酸来解决。尽管如此，抑制不是一个开关功能，目前的诱变研究表明，必须有一个C47独立的失活机制。实际结果是：发现了几乎完全抗性的突变体C47 L，其没有显示立体选择性的损失，这使得该变体作为有希望的催化剂。
The 2-deoxy-o-ribose-5-phosphate aldolase (DERA) is a synthetically attractive enzyme because of its ability to perform C-C-couplings stereoselectively, the enzyme uses acetaldehyde as nucleophile and thus produces true aldols rather than ketols, and may add two acetaldehyde molecules onto one electrophile. However, DERA produces crotonaldehyde as side reaction from acetaldehyde which is then an irreversible inhibitor forming a covalent Michael-adduct within the active site in particular with cysteine 47 (Dick et al., 2016). This inhibition can be resolved by mutating C47 to non-nucleophile amino acids. Still, the inhibition is not an on-off-feature and the present mutagenesis study illustrates that there must be a C47-independent inactivation mechanism. As a practical result: The virtually fully resistant mutant C47L was found, which shows no loss in stereoselectivity, this renders this variant as promising catalyst.