title: 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 with Gln, 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 ~2500-fold slower than those of wild-type MTOX. Wild-type MTOX forms a charge-transfer E(ox)·S complex with the redox-active anionic form of NMT. The E(ox)·S complex formed with Lys259Gln does not exhibit a charge-transfer band and is converted to a reduced enzyme·imine complex (EH(2)·P) at a rate 60-fold slower than that of wild-type MTOX. The mutant EH(2)·P complex contains the imine zwitterion and exhibits a charge-transfer band, a feature not observed with the wild-type EH(2)·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 EH(2)·P complex is 80-fold slower than k(cat). The mutant EH(2)·P complex dissociates 15-fold faster than k(cat,app). Consequently, EH(2)·P and free EH(2) 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Bruckner
authors.forename: Jennifer
authors.initials: J
authors.lastname: Winans
authors.forename: Marilyn Schuman
authors.initials: MS
authors.lastname: Jorns
mesh_terms: D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D005415:Flavins; D005973:Glutamine; C102761:SolA protein, E coli; D010089:Oxidoreductases, N-Demethylating; D008239:Lysine; D000409:Alanine