Characterization of dimethylsulfoxide, pyridine N-oxide, and trimethylamine N-oxide reductases of Escherichia coli

• Main Author: Simala-Grant, Joanne Lisa
• Format: Dissertation
• Language: English
• Subjects: chemistry, biochemistry
• ISBN: 9780612291096; 061229109X

E. coli dimethylsulfoxide reductase (DmsABC) is a trimeric iron-sulfur molybdoenzyme that allows respiratory growth on S- and N-oxides. Absorption and fluorescence spectroscopy was employed, and indicated DmsABC binds molybdopterin guanine dinucleotide (MGD).
A soluble form of DmsABC, missing the membrane anchor (DmsC), is found in the cytoplasm (DmsAB). Examination of the properties of DmsAB, and reconstitution of MGD into apo-DmsAB, confirmed the instability of DmsAB in comparison with DmsABC. These data implicated DmsC in the stabilization of DmsABC.
Determination of the correct initiating Met for DmsA indicated DmsA possesses a leader with a double-arginine consensus, suggested to be important for membrane targeting and translocation of a subset of redox cofactor containing proteins. Examination of DmsABC with truncated, deleted, or mutated double-arginine consensus leader, demonstrated the leader and consensus within, are essential for production of functional enzyme.
Open, closed, and nuclear magnetic resonance spectroscopic assays were examined for use with DmsABC. Investigation suggested dithionite was both a competitive and irreversible inhibitor. The kinetic constants for DmsABC were determined for both the electron donor, and electron acceptor portions of the reaction. This showed DmsABC's very broad electron acceptor specificity for S- and N-oxides and miscellaneous compounds. This was demonstrated to contrast trimethylamine N-oxide reductase (TorA), as it was shown to reduce only a few N-oxides.
Examination of the crystal structure of dimethylsulfoxide reductase from Rhodobacter sphaeroides (DMSOR), and comparison of the homologous DmsA and DMSOR sequences, revealed potential DmsA active site residues. Site-directed mutagenesis was employed to alter these residues in DmsA. Kinetic analysis demonstrated T148, A178, and R217 altered the electron acceptor K$\sb{\rm m},$ suggesting a role in substrate binding. G167 and Q179I decreased the k$\sb{\rm cat}$ for DMN, and abolished growth on Gly/DMSO, suggesting a role in electron transfer.
Examination of respiratory growth of E. coli not expressing DmsABC or TorA, indicated the presence of an additional previously unidentified anaerobically expressed energy conserving terminal reductase. It allowed for anaerobic growth on substituted pyridine N-oxides. The enzyme responsible, pyridine N-oxide reductase, was characterized, and appears to be a cytoplasmic molybdoenzyme.