|
Pavel D. Parshin, Ustina A. Martysuk, Denis L. Atroshenko, Anna V. Popinako, Svyatoslav S. Savin, Evgeny V. Pometun, Vladimir I. Tishkov, Anastasia A. Pometun
Study
of mechanism of coenzyme specificity of phenylacetone monooxygenase from Thermobifida
fusca by site-directed mutagenesis
Abstract
Abstract. Phenylacetone monooxygenase from Thermobifida
fusca (EC 1.14.13.92, PAMO) is a part of Baeyer–Villiger monooxygenases
family. It catalyzes oxidation of different aromatic ketones to corresponding
esters using NADPH as cofactor. In present work we analyzed cofactor-binding
center, selected residues which are potentially important for recognition of 2′-phosphate
group, followed by modeling amino acid substitutions which could result in
change of the enzyme cofactor specificity from NADPH to NADH. As result of
modeling the next amino acid changes were proposed – T218D, T218E, K336A,
K336R. Plasmids with genes of PAMO with proposed mutations were prepared by
site-directed mutagenesis. Mutant enzymes were expressed, purified and
characterized. New mutant PAMOs bound NADH but they did not catalyzed benzyl
acetone oxidation in presence of NADH and showed worse KM
values with NADPH. Values of catalytic constants of mutant enzymes are slightly
lower compared to wild type enzyme but deviations are in the range of
experimental errors.
Key words: phenylacetone
monooxygenase, Baeyer–Villiger monooxygenase, protein engineering,
site-directed mutagenesis, coenzyme specificity
Copyright (C) Chemistry Dept., Moscow State University, 2002
|
|
