1. Biosynthesis of topa quinone cofactor in bacterial amine oxidases. Solvent origin of C-2 oxygen determined by Raman spectroscopy.
- Author
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Nakamura N, Matsuzaki R, Choi YH, Tanizawa K, and Sanders-Loehr J
- Subjects
- Amine Oxidase (Copper-Containing) chemistry, Amino Acid Oxidoreductases chemistry, Cloning, Molecular, Copper pharmacology, Dihydroxyphenylalanine biosynthesis, Dihydroxyphenylalanine chemistry, Escherichia coli, Indicators and Reagents, Isotope Labeling methods, Methylamines, Oxygen Isotopes, Phenylhydrazines, Recombinant Proteins metabolism, Solvents, Spectrum Analysis, Raman, Water metabolism, Amine Oxidase (Copper-Containing) metabolism, Amino Acid Oxidoreductases metabolism, Arthrobacter enzymology, Coenzymes biosynthesis, Dihydroxyphenylalanine analogs & derivatives
- Abstract
Resonance Raman spectroscopy is an excellent technique for providing structural information on the 2,4, 5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper-containing amine oxidases. This technique has been used to investigate the copper- and O2-dependent biosynthesis of the TPQ cofactor in phenylethylamine oxidase (PEAO) and histamine oxidase from Arthrobacter globiformis. Incubation of the holoenzyme in H218O causes frequency shifts at 1684(-26) cm-1 in PEAO and at 1679(-28) cm-1 in histamine oxidase, allowing this feature to be assigned to the C=O stretch of a single carbonyl group at the C-5 position. When apoprotein is reacted with Cu(II) and O2 in the presence of H218O, the resultant holoproteins show increased shifts of -3 to -6 cm-1 in a number of other vibrational modes, particularly at 411 and 1397 cm-1. Because these small shifts persist when the H218O-regenerated protein is back-exchanged into H216O, they can be assigned to oxygen isotope substitution at the C-2 postion. No isotope shifts are observed when apoprotein is regenerated with Cu(II) in the presence of 18O2. Thus, it is concluded that the C-2 oxygen atom of TPQ originates from H2O rather than O2. The isotope dependence of the 1397-cm-1 mode allows it to be assigned to the C O moiety at the C-2 position, with its low frequency being indicative of only partial double bond character. Similar frequency shifts due to 18O at C-2 are observed in the resonance Raman spectra of H218O-regenerated PEAO after derivatization of the C-5 carbonyl with either p-nitrophenylhydrazine (-5 cm-1 at 480 cm-1) or methylamine (-5 cm-1 at 1301 cm-1). Taken together, these results indicate that the TPQ cofactor in the native enzyme has substantial electron delocalization between the C-2 and C-4 oxygens and that only the C-5 oxygen has predominantly C=O character.
- Published
- 1996
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