Journal article
Pleiotropic impact of a single lysine mutation on biosynthesis of and catalysis by N-methyltryptophan oxidase
Biochemistry (Easton), v 50(22), pp 4949-4962
07 Jun 2011
PMID: 21526853
Featured in Collection : UN Sustainable Development Goals @ Drexel
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.
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Details
- Title
- Pleiotropic impact of a single lysine mutation on biosynthesis of and catalysis by N-methyltryptophan oxidase
- Creators
- Robert C Bruckner - Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USAJennifer WinansMarilyn Schuman Jorns
- Publication Details
- Biochemistry (Easton), v 50(22), pp 4949-4962
- Publisher
- American Chemical Society; Washington, DC
- Grant note
- R01 GM031704 / NIGMS NIH HHS R01 GM031704-25 / NIGMS NIH HHS GM 31704 / NIGMS NIH HHS
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:000290978200012
- Scopus ID
- 2-s2.0-79958065906
- Other Identifier
- 991014877786504721
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- Biochemistry & Molecular Biology