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

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 Gin, Ala, or Met blocks (>95%) covalent flavin incorporation in vivo. The mutant apoproteins can be reconstituted with FAD. Apparent turnover rates (k(cat,app)) of the reconstituted enzymes are similar to 2500-fold slower than those of wild-type MTOX. Wild-type MTOX forms a charge-transfer E-ox center dot S complex with the redox-active anionic form of NMT. The E-ox center dot S complex formed with Lys259Gln does not exhibit a charge-transfer band and is converted to a reduced enzyme center dot imine complex (EH2 center dot P) at a rate 60-fold slower than that of wild-type MTOX. The mutant EH2 center dot P complex contains the imine zwitterion and exhibits a charge-transfer band, a feature not observed with the wild-type EH2 center dot 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 center dot P complex is 80-fold slower than k(cat). The mutant EH2 center dot P complex dissociates 15-fold faster than k(cat,app). Consequently, EH2 center dot 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.