Role of conserved Asn-Tyr-Asp-Tyr sequence in bacterial copper/2,4, 5-trihydroxyphenylalanyl quinone-containing histamine oxidase.
Copper amine oxidase contains a covalently bound quinonoid cofactor, 2,4,5-trihydroxyphenylalanyl quinone (TPQ), which is synthesized by post-translational modification of a specific tyrosyl residue occurring in the highly conserved sequence, Asn-Tyr-(Asp/Glu)-Tyr. To elucidate the role(s) of the conserved sequence in the biogenesis of TPQ, each of the corresponding residues at positions 401-404 in the recombinant histamine oxidase from Arthrobacter globiformis 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. Furthermore, when Tyr-404 was changed to Phe, TPQ was formed swiftly on incubation with copper ions, but the TPQ enzyme exhibited very low activity with altered substrate specificity. 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.
Choi
Y H
YH
Division of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567, Japan.
Matsuzaki
R
R
Suzuki
S
S
Tanizawa
K
K
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D017124:Conserved Sequence; D003300:Copper / Q000378:metabolism; D018360:Crystallography, X-Ray; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D010101:Oxygen Consumption; D010659:Phenylhydrazines / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D013054:Spectrophotometry, Atomic
C086758:2,4,5-trihydroxyphenylalanine quinone; D010659:Phenylhydrazines; C030299:phenylhydrazine; D004295:Dihydroxyphenylalanine; D003300:Copper; D006631:Amine Oxidase (Copper-Containing)