Controlling the regioselectivity and stereospecificity of FAD-dependent polyamine oxidases with the use of amine-attached guide molecules as conformational modulators

Abstract

Enzymes generally display strict stereospecificity and regioselectivity for their substrates. Here by using FAD-dependent human acetylpolyamine oxidase (APAO), human spermine (Spm) oxidase (SMOX) and yeast polyamine oxidase (Fms1), we demonstrate that these fundamental properties of the enzymes may be regulated using simple guide molecules, being either covalently attached to polyamines or used as a supplement to the substrate mixtures. APAO, which naturally metabolizes achiral N 1 -acetylated polyamines, displays aldehyde-controllable stereospecificity with chiral 1-methylated polyamines, like (R)- and (S)-1-methylspermidine (1,8-diamino-5-azanonane) (1-MeSpd). Among the novel N 1 -acyl derivatives of MeSpd, isonicotinic acid (P4) or benzoic acid (Bz) with (R)-MeSpd had K m of 3.6 + − 0.6/1.2 + − 0.7 μM and k cat of 5.2 + − 0.6/4.6 + − 0.7 s −1 respectively, while N 1 -AcSpd had K m 8.2 + − 0.4 μM and k cat 2.7 + − 0.0 s −1 . On the contrary, corresponding (S)-MeSpd amides were practically inactive (k cat < 0.03 s −1 ) but they retained micromole level K m for APAO. SMOX did not metabolize any of the tested compounds (k cat < 0.05 s −1 ) that acted as non-competitive inhibitors having K i ≥ 155 μM for SMOX. In addition, we tested (R,R)-1,12-bis-methylspermine (2,13-diamino-5,10-diazatetradecane) (R,R)-(Me 2 Spm) and (S,S)-Me 2 Spm as substrates for Fms1. Fms1 preferred (S,S)- to (R,R)-diastereoisomer, but with notably lower k cat in comparison with spermine. Interestingly, Fms1 was prone to aldehyde supplementation in its regioselectivity, i.e. the cleavage site of spermidine. Thus, aldehyde supplementation to generate aldimines or N-terminal substituents in polyamines, i.e. attachment of guide molecule, generates novel ligands with altered charge distribution changing the binding and catalytic properties with polyamine oxidases. This provides means for exploiting hidden capabilities of polyamine oxidases for controlling their regioselectivity and stereospecificity.