title: The structure of maize polyamine oxidase K300M mutant in complex with the natural substrates provides a snapshot of the catalytic mechanism of polyamine oxidation.
abstract: Polyamine oxidases are FAD-dependent enzymes catalyzing the oxidation of polyamines at the secondary amino groups. Zea mays PAO (ZmPAO) oxidizes the carbon on the endo-side of the N5-nitrogen of spermidine (Spd) and spermine (Spm). The structure of ZmPAO revealed that the active site is formed by a catalytic tunnel in which the N5 atom of FAD lies in close proximity to the K300 side chain, the only active-site residue conserved in all PAOs. A water molecule, (HOH309), is hydrogen-bound to the amino group of K300 and mutation of this residue results in a 1400-fold decrease in the rate of flavin reduction. The structural studies on the catalytically impaired ZmPAO-K300M mutant described here show that substrates are bound in an 'out-of-register' mode and the HOH309 water molecule is absent in the enzyme-substrate complexes. Moreover, K300 mutation brings about a 60 mV decrease in the FAD redox potential and a 30-fold decrease in the FAD reoxidation rate, within a virtually unaltered geometry of the catalytic pocket. Taken together, these results indicate that the HOH309-K300 couple plays a major role in multiple steps of ZmPAO catalytic mechanism, such as correct substrate binding geometry as well as FAD reduction and reoxidation kinetics.
authors.affiliation: Department of Science and Biomedical Technology, University of L'Aquila, Italy.
authors.forename: Annarita
authors.initials: A
authors.lastname: Fiorillo
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Federico
authors.forename: Fabio
authors.initials: F
authors.lastname: Polticelli
authors.forename: Alberto
authors.initials: A
authors.lastname: Boffi
authors.forename: Franco
authors.initials: F
authors.lastname: Mazzei
authors.forename: Massimo
authors.initials: M
authors.lastname: Di Fusco
authors.forename: Andrea
authors.initials: A
authors.lastname: Ilari
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Tavladoraki
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013095:Spermidine / Q000378:metabolism; D013096:Spermine / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*; D000097667:Polyamine Oxidase
chemical_list: D000587:Oxidoreductases Acting on CH-NH Group Donors; D010940:Plant Proteins; D011073:Polyamines; D013095:Spermidine; D013096:Spermine; D000097667:Polyamine Oxidase