The structure of maize polyamine oxidase K300M mutant in complex with the natural substrates provides a snapshot of the catalytic mechanism of polyamine oxidation.

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.

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