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 pos t-translational modification of a specific tyrosyl residue occurring i n the highly conserved sequence, Asn-Tyr-(Asp/Glu)-Tyr. To elucidate t he role(s) of the conserved sequence in the biogenesis of TPQ, each of the corresponding residues at positions 401-404 in the recombinant hi stamine oxidase from Arthrobacter globiformis has been replaced with o ther amino acids by site-directed mutagenesis. When Asn 401 was change d to Asp or Gln, the rate of TPQ formation by copper-dependent self-pr ocessing 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-40 3 could be replaced by Glu without changes in the rate of TPQ formatio n, whereas its replacement by Asn led to a marked decrease. Furthermor e, when Tyr-404 was changed to Phe, TPQ was formed swiftly on incubati on with copper ions, but the TPQ enzyme exhibited very low activity wi th altered substrate specificity. These results collectively indicate that a very rigorous structural motif is required for efficient format ion of TPQ and for the catalytic activity in the active site of copper amine oxidases.