Structural Bases for Inhibitor Binding and Catalysis in Polyamine Oxidase^,

Abstract

Polyamine oxidase (PAO) carries out the FAD-dependent oxidation of the secondary amino groups of spermidine and spermine, a key reaction in the polyamine catabolism. The active site of PAO consists of a 30 A long U-shaped catalytic tunnel, whose innermost part is located in front of the flavin ring. To provide insight into the PAO substrate specificity and amine oxidation mechanism, we have investigated the crystal structure of maize PAO in the reduced state and in complex with three different inhibitors, guazatine, 1,8-diaminooctane, and N(1)-ethyl-N(11)-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm). In the reduced state, the conformation of the isoalloxazine ring and the surrounding residues is identical to that of the oxidized enzyme. Only Lys300 moves away from the flavin to compensate for the change in cofactor protonation occurring upon reduction. The structure of the PAO.inhibitor complexes reveals an exact match between the inhibitors and the PAO catalytic tunnel. Inhibitor binding does not involve any protein conformational change. Such lock-and-key binding occurs also in the complex with CHENSpm, which forms a covalent adduct with the flavin N5 atom. Comparison of the enzyme complexes hints at an "out-of-register" mechanism of inhibition, in which the inhibitor secondary amino groups are not properly aligned with respect to the flavin to allow oxidation. Except for the Glu62-Glu170 pair, no negatively charged residues are involved in the recognition of substrate and inhibitor amino groups, which is in contrast to other polyamine binding proteins. This feature may be exploited in the design of drugs specifically targeting PAO.

Mechanistic Studies of Mouse Polyamine Oxidase with N1,N12-Bisethylspermine as a Substrate

“…No such longwavelength absorbance is detected upon reduction of polyamine oxidase by BESPM; a simple explanation is that the product dissociates from the reduced enzyme rapidly. Consistent with this interpretation, the structure of maize polyamine oxidase reduced by spermine shows that the product is not in the active site (13). The oxidative half-reaction of mouse polyamine oxidase can be described as a simple second-order reaction of reduced polyamine oxidase with oxygen with the second-order rate constant k ox to complete the catalytic cycle.…”

“…The mammalian polyamine oxidases have undergone little mechanistic study. The structure of a maize polyamine oxidase has been described with a number of inhibitors (13,14), establishing the enzyme as a homologue of mitochondrial monoamine oxidase, and preliminary kinetic studies with spermine as a substrate have been reported for that enzyme (15). However, the plant enzymes prefer spermine and spermidine as substrates instead of acetylated polyamines (16), so they are better described as spermine oxidases than polyamine oxidases.…”

Structure-Function Relationships in Flavoenzyme-dependent Amine Oxidations

“…In particular, Lys-300 of PAO is bridged to the N5 atom of the flavin through a water molecule. Binding studies with inhibitors (19) have shown that this residue participates in catalysis by compensating for the change in the flavin protonation state (i.e. the addition of a hydrogen atom to the N5-position, which occurs on cofactor reduction).…”

“…On the basis of the structures of enzyme-inhibitor complexes (3,19), models of the bound substrates in the active sites have been proposed with reference to spermine for PAO (Figs. 1B and 3A) and to benzylamine for MAO B (Figs.…”

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine

“…The recombinant protein expression of the mammalian APAO and SMO in different heterologous systems provided the basis for a deeper understanding of the structure/function relationships of these enzymes [11][12][13][14][15][16] . At present, only the three-dimensional structures of two members of the polyamine oxidase family are available, namely the maize polyamine oxidase (ZmPAO 17,18 ) and the yeast polyamine oxidase (FMS1 19 ). Molecular modeling of mouse SMO based on the three-dimensional structure of ZmPAO and sequence analysis of animal polyamine oxidases indicated that the general features of the ZmPAO threedimensional structure are conserved in the mammalian enzymes 11,20,21 .…”