Pleiotropic Impact of a Single Lysine Mutation on Biosynthesis of and Catalysis by N-Methyltryptophan Oxidase

Abstract

N-Methyltryptophan oxidase (MTOX) contains covalently bound FAD. N-Methyltryptophan binds in a cavity above the re-face of the flavin ring. Lys259 is located above the opposite, si-face. Replacement of Lys259 by Gln, Ala, or Met blocks (>95%) covalent flavin incorporation in vivo. The mutant apoproteins can be reconstituted with FAD. Apparent turnover rates (kcat app) of the reconstituted enzymes are about 2500-fold slower than wild-type MTOX. Wild-type MTOX forms a charge-transfer Eox*S complex with the redox-active anionic form of NMT. The Eox*S complex formed with Lys259Gln does not exhibit a charge-transfer band and is converted to a reduced enzyme*imine complex (EH2*P) at a 60-fold slower rate than wild-type MTOX. The mutant EH2*P complex contains the imine zwitterion and exhibits a charge-transfer band, a feature not observed with the wild-type EH2*P complex. Reaction of reduced Lys259Gln with oxygen is 2500-fold slower than reduced wild-type MTOX. The latter reaction is unaffected by the presence of bound product. Dissociation of the wild-type EH2*P complex is 80-fold slower than kcat. The mutant EH2*P complex dissociates 15-fold faster than kcat app. Consequently, EH2*P and free EH2 are the species that react with oxygen during turnover of wild-type and mutant enzyme, respectively. The results show that: (i) Lys259 is the site of oxygen activation in MTOX and also plays a role in holenzyme biosynthesis and N-methyltryptophan oxidation; (ii) MTOX contains separate active sites for N-methyltryptophan oxidation and oxygen reduction on opposite faces of the flavin ring.

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 . …”

Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2

“…Binding superoxide is equivalent to increasing the potential of the O 2 /superoxide couple, helping to lower the energy barrier of the rate-determining first step. There are many examples of large decreases in O 2 -reactivity when a positive residue near the flavin is neutralized, either by mutagenesis (e.g., fructosamine oxidase, monomeric sarcosine oxidase, tryptophan oxidase) or by deprotonation at high pH values (e.g., glucose oxidase) . For example, a protonated histidine residue positioned near the N5 of FAD in glucose oxidase enhanced reactivity with oxygen by ∼10 000-fold .…”

“…Such a relationship between structure and reactivity is curiously unreliable. NicA2 has a lysine poised close to N5 in a position similar to lysines in several bona fide oxidases where the lysine has been demonstrated to be critical to reactivity. − Obviously, the presence of this lysine in NicA2 does not enhance O 2 -reactivity, which is more than 100-fold lower than aqueous flavin. A Class 2 dihydroorotate dehydrogenase with a similarly positioned lysine reacts with O 2 as a slow oxidase (∼10 4 M –1 s –1 ); neutralizing it by mutation did not alter O 2 reactivity, again demonstrating a surprising insensitivity of O 2 reactivity to a nearby positive charge .…”

The enigmatic reaction of flavins with oxygen

“…also resulted in large (from 550-to 2500-fold) decreases in the reoxidation rate constants of the enzyme bound reduced flavins [20,21]. A positive charge around the flavin binding site was also shown to be a key feature for enhancing the oxygen reactivity in choline oxidase, illustrating an interesting variation on this theme [22].…”