Mechanistic studies of the role of a conserved histidine in a mammalian polyamine oxidase

Abstract

Polyamine oxidases are peroxisomal flavoproteins that catalyze the oxidation of an endo carbon nitrogen bond of N1-acetylspermine in the catabolism of polyamines. While no structure has been reported for a mammalian polyamine oxidase, sequence alignments of polyamine oxidizing flavoproteins identify a conserved histidine residue. Based on the structure of a yeast polyamine oxidase, S. cerevisiae Fms1, this residue has been proposed to hydrogen bond to the reactive nitrogen in the polyamine substrate. The corresponding histidine in mouse polyamine oxidase, His64, has been mutated to glutamine, asparagine, and alanine to determine if this residue plays a similar role in the mammalian enzymes. The kinetics of the mutant enzymes were examined with N1-acetylspermine and the slow substrates spermine and N, N′-dibenzyl-1,4-diaminobutane. On average the mutations result in a decrease of ~15-fold in the rate constant for amine oxidation. Rapid -reaction kinetic analyses established that amine oxidation is rate-limiting with spermine as substrate for the wild -type and mutant enzymes and for the H64N enzyme with N1-acetylspermine as substrate. The kcat/KO2 value was unaffected by the mutations with N1-acetylspermine as substrate, but decreased ~55-fold with the two slower substrates. The results are consistent with this residue assisting in properly positioning the amine substrate for oxidation.

The Structure of Murine N1-Acetylspermine Oxidase Reveals Molecular Details of Vertebrate Polyamine Catabolism

“…Lys 315 has been implicated in the oxidative half-reaction, 21 and similar roles have been proposed for the corresponding residues in SMO (Lys 367) 22 and ZmPAO (Lys 300). 23 The site of catalysis is located in the junction between the two subpockets where the catalytically important His 64 24 and the highly conserved Tyr 430 5 line a narrow groove. While the electrostatic character of the hydrophobic subpocket is neutral, the other subpocket holds two distinguishable patches of negative charge.…”

“…A histidine within the binding site, which is highly conserved across many polyamine oxidases, has been suggested to play a role in both directing the substrate to the catalytic site (His 64 in APAO) 24 and participating in a hydrogen bond network that is important for maintaining the overall active site structure (His 67−Asn 195−Asp 94 in Fms1). 33 Analysis of the APAO− substrate complex shows that His 64 is indeed positioned close enough to interact with N5 of N 1 -acetylspermine through a hydrogen bond at physiological pH.…”

Toxicity of Polyamines and Their Metabolic Products

“…This review will focus on oxidases present in mammalian tissues. (There are several useful reviews on the plant and yeast polyamine oxidases and the molecular evolution of the family of polyamine oxidases. ,− ) Unfortunately, the nomenclature of these enzymes is confusing The mechanistic aspects of amine oxidation are well understood, and they are best characterized in terms of their cofactors and preferred substrates. Detailed mechanistic and structural studies have shown that these oxidases can be classified as Cu 2+ -containing and FAD-requiring.…”

A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines

“…1). Among them, His82 has an important role in substrate binding (Tavladoraki et al, 2011;Adachi et al, 2012;Tormos et al, 2012) and is conserved in most PAOs involved in polyamine back-conversion, such as, for example, MmAPAO, yeast FMS1, AtPAO2, AtPAO3, and AtPAO4, but not in AtPAO1 (Fig. 1).…”