Abstract
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.
Highlights
We reported recently that the inactive, copper-free form of the recombinant histamine oxidase from Arthrobacter globiformis was capable of forming trihydroxyphenylalanyl quinone (TPQ) when incubated aerobically with copper ion (Choi et al, 1995)
The essential role of Tyr-402 as the precurtification of 2,4,5-trihydroxyphenylalanyl quinone (TPQ)1 as sor to TPQ was confirmed. The roles of these conserved resithe cofactor of the bovine serum enzyme (Janes et al, 1990), dues in the biogenesis of TPQ in copper amine oxidases are TPQ has been shown to be the common cofactor in all members discussed on the basis of the recently determined x-ray crysof the copper amine oxidase family (Janes et al, 1992; Knowles tallographic structure of the Escherichia coli amine oxidase and Dooley, 1994)
The stalk domain (D1) is absent from some copper amine oxidases including histamine oxidase from A. globiformis studied here, but the cap consisting of two small peripheral domains (D2 and D3) and a large core with the active site is conserved throughout the copper amine oxidase family (Parsons et al, 1995)
Summary
Construction of Improved Expression Plasmid—To achieve higher expression of histamine oxidase than that of pTrc99HAO constructed previously (Choi et al, 1995), a hybrid vector (named pUT) was con-. The wild-type and mutant enzymes were purified to homogeneity in the copper-free form using the procedure reported previously (Choi et al, 1995). The copper-containing form of the wild-type and mutant enzymes were prepared by overnight incubation at 4 °C with an excess of [5–10] mol equivalents of CuSO4 over the enzyme subunit. They were dialyzed thoroughly against 50 mM HEPES, pH 6.8, containing 3 mM EDTA, followed by dialysis against buffer alone to remove the unbound copper. Atomic Absorption Analysis—The copper contents of the enzyme proteins were analyzed with a Nippon Jarrell-Ash AA-880 mark II atomic absorption spectrophotometer (acetylene/air flame) at 324.8 nm
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