Correlation of active site metal content in human diamine oxidase with trihydroxyphenylalanine quinone cofactor biogenesis .
Copper-containing amine oxidases (CAOs) require a protein-derived topaquinone cofactor (TPQ) for activity. TPQ biogenesis is a self-processing reaction requiring the presence of copper and molecular oxygen. Recombinant human diamine oxidase (hDAO) was heterologously expressed in Drosophila S2 cells, and analysis indicates that the purified hDAO contains substoichiometric amounts of copper and TPQ. The crystal structure of a complex of an inhibitor, aminoguanidine, and hDAO at 2.05 Å resolution shows that the aminoguanidine forms a covalent adduct with the TPQ and that the site is ∼75% occupied. Aminoguanidine is a potent inhibitor of hDAO with an IC(50) of 153 ± 9 nM. The structure indicates that the catalytic metal site, normally occupied by copper, is fully occupied. X-ray diffraction data recorded below the copper edge, between the copper and zinc edges, and above the zinc edge have been used to show that the metal site is occupied approximately 75% by copper and 25% by zinc and the formation of the TPQ cofactor is correlated with copper occupancy.
McGrath
Aaron P
AP
School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia.
Caradoc-Davies
Tom
T
Collyer
Charles A
CA
Guss
J Mitchell
JM
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000378:metabolism*; D016227:Benzoquinones; D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives; D006801:Humans; D008670:Metals; D011809:Quinones / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D014961:X-Ray Diffraction; D015032:Zinc
D016227:Benzoquinones; D008670:Metals; D011809:Quinones; D011994:Recombinant Proteins; C004532:quinone; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); D015032:Zinc