单加氧酶
化学
环己酮
半胱氨酸
热稳定性
酶
突变体
蛋氨酸
立体化学
硫茴香醚
生物化学
氨基酸
细胞色素P450
催化作用
基因
作者
Diederik J. Opperman,Manfred T. Reetz
出处
期刊:ChemBioChem
[Wiley]
日期:2010-11-15
卷期号:11 (18): 2589-2596
被引量:101
标识
DOI:10.1002/cbic.201000464
摘要
Baeyer-Villiger monooxygenases (BVMOs) catalyze the conversion of ketones and cyclic ketones into esters and lactones, respectively. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is known to show an impressive substrate scope as well as exquisite chemo-, regio-, and enantioselectivity in many cases. Large-scale synthetic applications of CHMO are hampered, however, by the instability of the enzyme. Oxidation of cysteine and methionine residues contributes to this instability. Designed mutations of all the methionine and cysteine residues in the CHMO wild type (WT) showed that the amino acids labile towards oxidation are mostly either surface-exposed or located within the active site, whereas the two methionine residues identified for thermostabilization are buried within the folded protein. Combinatorial mutations gave rise to two stabilized mutants with either oxidative or thermal stability, without compromising the activity or stereoselectivity of the enzyme. The most oxidatively stabilized mutant retained nearly 40 % of its activity after incubation with H(2)O(2) (0.2 M), whereas the wild-type enzyme's activity was completely abolished at concentrations as low as 5 mM H(2)O(2). We propose that oxidation-stable mutants might well be a "prerequisite" for thermostabilization, because laboratory-evolved thermostability in CHMO might be masked by a high degree of oxidation instability.
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