Abstract
The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.
Highlights
From the $Department of Chemistry, Uniuersity of California, Berkeley, California 94720, the §Beckman Center, Stanford University, Stanford, California94305, and the lDepartmentof Chemistry, AmherstCollege, Amherst, Massachusetts01002 the cloning of a bovine serum amine oxidase (BSAO) gene has not yet been accomplished, the yeast amine oxidase (YAO) gene from Hansenula polym o r p h has been cloned and sequenced [2]
Comparison of resonance Ramanspectra for this amino acid residue in the polypeptidebackbone
In in the YAO DNA allows us to conclude that tyrosine is the precursor to topa quinone in eukaryotes
Summary
The intercept of a copper standard curve was significantly elevated in the presenceof YAO, relative to a standard curve in the absence of added protein (Fig. 2, inset). The elution profile of a YAO digest is shown inFig. 3, revealing a major chromophoric (Fig. 3A) and radiolabeled peak (Fig. 3C), which elutes near the end of the gradient This pewakas further purified by HPLC, prior to analysis.:’ Peptide sequencing has indicated a heptapeptide of the following sequence, Val-Ala-Asn-X-Glu-TyrVal. In order to identify the nature of the unknown, a resonance Raman spectrum of the peptide was compared with a spectrum of a model compound, the hydantoinof topa quinone which has beenderivatized withphenylhydrazine [1].these spectra were found to be essentially identicalwith regard topeak frequencies andintensities, providing clear-cut evidence for the presence of topa quinone at the YAO active site. Four nmol of pure, radiolabeled peptide were isolated for a final yield of 15%
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