Max-Planck-Institut für Kohlenforschung, Department of Synthetic Organic Chemistry, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany.
Chembiochem. 2010 Dec 10;11(18):2589-96. doi: 10.1002/cbic.201000464.
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.
拜耳-维利格单加氧酶（BVMOs）分别催化酮和环酮转化为酯和内酯。众所周知，来自不动杆菌属 NCIMB 9871 的环己酮单加氧酶（CHMO）在许多情况下具有令人印象深刻的底物范围以及出色的化学、区域和对映选择性。然而，CHMO 的大规模合成应用受到酶不稳定的限制。半胱氨酸和蛋氨酸残基的氧化导致了这种不稳定性。对 CHMO 野生型（WT）中所有蛋氨酸和半胱氨酸残基的设计突变表明，易氧化的氨基酸大多位于表面或活性部位内，而确定用于热稳定性的两个蛋氨酸残基则埋藏在折叠的蛋白质中。组合突变产生了两个具有氧化或热稳定性的稳定突变体，而不影响酶的活性或立体选择性。最具氧化稳定性的突变体在与 H(2)O(2)（0.2 M）孵育后保留了近 40%的活性，而野生型酶的活性在低至 5 mM H(2)O(2)的浓度下就完全被抑制。我们提出，氧化稳定的突变体很可能是热稳定性的“先决条件”，因为 CHMO 中的实验室进化的热稳定性可能被高度氧化不稳定性所掩盖。
