| Research Abstract |
Copper amine oxidase catalyzes oxidative deamination of various biogenic amines, occurring widely in microorganisms, plants and animals. Besides copper, the enzyme contains a recently identified quinonoid cofactor, 2,4,5-trihydroxyphenylalanyl quinone (TPQ), covalently bound to the protein. In this study, the genes coding for phenylethylamine oxidase and histamine oxidase have been cloned from a Coryne-form bacterium Arthrobacter globiformis and sequenced. Using the recombinant apoenzyme overproduced in E.coil cells, the TPQ cofactor has been demonstrated to be produced from a specific tyrosyl residue by self-processing of the protein upon binding divalent copper ions. The process of TPQ formation has been investigated in detail by UV-VIS,CD,ESR,and resonance Raman spectroscopies to show that the cupric ion bound to the apoenzyme is first reduced to a cuprous ion, that the cuprous ion likely activates dioxygen for oxidative modification of the precursor tyrosyl residue, that a semiquin
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one radical intermediate is formed during the TPQ formation, and that the carbonyl C-2 oxygen of TPQ is derived from solvent. To elucidate the role(s) of the conserved sequence Ans-Tyr-Asp/Glu-Tyr in the biogenesis of TPQ,each of the corresponding residues at positions 401-404 in the recombinant histamine oxidase has been replaced with other amino acids by site-directed mutagenesis. When Asn-401 was changed to Asp or Gln, the rate of TPQ formation by copper-dependent self-processing was 10^3-to 10^4-fold slower than in the wild-type enzyme. When Tyr-402 was replaced by Phe, TPQ was not formed at all, showing that Tyr-402 is essential as the precursor to TPQ.In contrast, Asp-403 could be replaced by Glu without changes in the rate of TPQ formation, whereas its replacement by Asn led to a marked decrease. These results collectively indicate that a very rigorous structural motif is required for efficient formation of TPQ and for the catalytic activity in the active site of copper amine oxidases. The crystal structures phenylethylamine oxidase have been determined and refined for the inactive apo and active holo forms both at 2.2 resolution. Significant differences between the two forms are limited to the active site (positions and orientations of the precursor Tyr and TPQ and a His residue binding copper, suggesting that there occurrs no large conformational changes of the protein during the TPQ biogenesis. Less
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