ИСТИНА

Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a Bayer-Williger monooxygenase (BWMO) capable of oxidizing aldehydes and ketones to esters. In comparison with most of BWMOs PAMO has high temperature and chemical stability, which makes it promising enzyme for use in real biotechnology processes. In spite of listed advantages, wide usage of PAMO alike with other monooxygenases is obstructed by high cost of required NADPH. There is an efficient way to solve the problem - to create a NADPH regeneration system by another enzyme. For example, formate dehydrogenase (FDH). Although there are several NADPH-dependent FDHs, most of them use NADH. Therefore, it would be much more convenient if PAMO used the non-phosphorylated form (NADH) as a coenzyme.The presence of 3D structure of PAMO in the PDB databank made it possible to use computational modeling methods and select two amino acid residues (T218 and K336) presumably involved in phosphate group binding in active site of PAMO. Moreover, change of similar amino acid residues led to a change in the enzyme specificity towards NADH according to data on related Bayer- Villiger monooxygenases. Here we report results of structure modeling and properties of obtained mutant enzymes. To prepare mutant PAMO variants an enzyme with increased expression level and stability was used.The reported study was funded by Russian Foundation for Basic Research according to the research project № 20-34-90120