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

Authors:
Publisher website
Google Scholar
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 with 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 ∼2500-fold slower than those of 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 rate 60-fold slower than that of 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 that of 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 the 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 holoenzyme biosynthesis and N-methyltryptophan oxidation and (ii) MTOX contains separate active sites for N-methyltryptophan oxidation and oxygen reduction on opposite faces of the flavin ring.

This publication has 43 references indexed in Scilit: