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Pleiotropic impact of a single lysine mutation on biosynthesis of and catalysis by N-methyltryptophan oxidase
Journal article   Open access   Peer reviewed

Pleiotropic impact of a single lysine mutation on biosynthesis of and catalysis by N-methyltryptophan oxidase

Robert C Bruckner, Jennifer Winans and Marilyn Schuman Jorns
Biochemistry (Easton), v 50(22), pp 4949-4962
07 Jun 2011
DOI: https://doi.org/10.1021/bi200349m
PMID: 21526853
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1021/bi200349mView
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Abstract

Oxidoreductases, N-Demethylating - genetics Escherichia coli - enzymology Oxidation-Reduction Models, Molecular Escherichia coli Proteins - metabolism Oxidoreductases, N-Demethylating - chemistry Lysine - genetics Alanine - genetics Escherichia coli - genetics Escherichia coli - metabolism Glutamine - genetics Escherichia coli Proteins - genetics Lysine - metabolism Catalysis Kinetics Mutation Escherichia coli Proteins - chemistry Lysine - chemistry Oxidoreductases, N-Demethylating - metabolism Binding Sites Flavins - metabolism
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.

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Web of Science research areas
Biochemistry & Molecular Biology
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