13C, 15N, and 31P NMR studies on 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans.
The interaction between the apoprotein of 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans and the prosthetic group FAD has been investigated by 13C, 15N, and 31P NMR techniques. The FAD prosthetic group was selectively enriched in 13C and 15N isotopes by adding isotopically labeled riboflavin derivatives to the growth medium of riboflavin-requiring mutant cells. In the oxidized state the chemical shift of the C(7) and C(8) atoms indicates that the xylene moiety of the isoalloxazine ring is embedded in a hydrophobic environment. The polarization of the isoalloxazine ring as a whole is, however, much more comparable to that of free flavin in a polar and protic environment than to free flavin in an apolar environment. The polarization of the ring system can be ascribed to strong hydrogen bonds between the apoprotein and the two carbonyl groups. The binding of the competitive inhibitor, 6-hydroxy-D-nicotine, influences the resonances of the C(4a) and the N(5) atoms strongly. It is suggested that these shifts are due to a strong hydrogen-bonding interaction between the N(5) atom and the inhibitor. On reduction all resonances, except those of the C(10a) and the N(1) atoms, shift upfield, indicating the increased electron density in the ring system. In the dithionite-reduced enzyme, the ring system is bent at the N(5) position. Due to the bending of the N(5) atom and the sp2 hybridized N(10) atom, electron density from the N(10) atom is reallocated at the C(4) carbonyl group. In contrast, in the substrate-reduced enzyme the N(5) atom is almost completely sp2 hybridized, yielding a rather planar isoalloxazine ring.(ABSTRACT TRUNCATED AT 250 WORDS)
Pust
S
S
Biochemisches Institut der Albert-Ludwigs-Universität, Freiburg, FRG.
Vervoort
J
J
Decker
K
K
Bacher
A
A
Müller
F
F
D001173:Arthrobacter / Q000201:enzymology*; D002247:Carbon Isotopes; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009587:Nitrogen Isotopes; D010758:Phosphorus; D013053:Spectrophotometry
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010758:Phosphorus