Oxygen Reactivity in Flavoenzymes: Context Matters

Abstract

Many flavoenzymes – oxidases and monooxygenases – react faster with oxygen than free flavins do. There are many ideas on how enzymes cause this. Recent work has focused on the importance of a positive charge near N5 of the reduced flavin. Fructosamine oxidase has a lysine near N5 of its flavin. We measured a rate constant of 1.6 × 105 M−1s−1 for its reaction with oxygen. The Lys276Met mutant reacted with a rate constant of 291 M−1s−1, suggesting an important role for this lysine in oxygen activation. The dihydroorotate dehydrogenases from E. coli and L. lactis also have a lysine near N5 of the flavin. They react with O2 with rate constants of 6.2 × 104 M−1s−1 and 3.0 × 103 M−1s−1, respectively. The Lys66Met and Lys43Met mutant enzymes react with rate constants that are nearly the same as the wild-type enzymes, demonstrating that simply placing a positive charge near N5 of the flavin does not guarantee increased oxygen reactivity. Our results show that the lysine near N5 does not exert an effect without an appropriate context; evolution did not find only one mechanism for activating the reaction of flavins with O2.

Engineering of pyranose 2-oxidase for modified oxygen reactivity

“…while dehydrogenase activity was maintained or only slightly reduced with DCPIP or 1,4-BQ. Oxygen-reacting flavoenzymes often carry positively charged groups near N5 of the isoalloxazine to enhance the reaction with oxygen [31], and to date histidine, lysine or a charged product formed at the active site have been shown to act as such an electrostatic catalyst [33]. At first glance our results seem to contradict other reports, since the introduction of a positive charge greatly reduced reactivity with oxygen.…”

“…The environment around the C4a-N5 locus, which is directly involved in the reaction with oxygen, is discussed to be the crucial area affecting oxidase activity [28]. A positive charge around the flavin reaction site, which can be provided by amino acid residues [15,26,[29][30][31][32] or bound substrate and/or product [33][34][35][36], can influence this. Channels leading from the surface to the active site may affect oxidase reactivity [37,38].…”

Relative Timing of Hydrogen and Proton Transfers in the Reaction of Flavin Oxidation Catalyzed by Choline Oxidase

“…It complements previous studies of choline oxidase and other oxidases in which the mechanism for O 2 activation was probed with mechanistic tools. 4,16,17,[36][37][38]42 This study also provides a framework for future studies of choline oxidase that will be aimed at the elucidation of the contribution of active site residues toward reduction of O 2 . …”

Development of Halogenase Enzymes for Use in Synthesis

“…242,384 Knowledge of the mechanisms of oxygen activation by the monooxygenases could also be used to promote formation and stabilisation of this key intermediate. 385 It is possible however that a lack of productivity with the Fl-Hals could be due largely to the fact that the rate-determining step (electrophilic aromatic substitution) is very energetically demanding and therefore engineering could be focussed upon stabilisation of the Wheland intermediate or increasing electrophilicity of the halogenating species. The outcomes from the rational methods to improve the oxidative and thermostability of BVMO could also be useful to FlHal engineering.…”