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 N1-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 N1-acyl derivatives of MeSpd, isonicotinic acid (P4) or benzoic acid (Bz) with (R)-MeSpd had Km of 3.6 ± 0.6/1.2 ± 0.7 µM and kcat of 5.2 ± 0.6/4.6 ± 0.7 s−1 respectively, while N1-AcSpd had Km 8.2 ± 0.4 µM and kcat 2.7 ± 0.0 s−1. On the contrary, corresponding (S)-MeSpd amides were practically inactive (kcat < 0.03 s−1) but they retained micromole level Km for APAO. SMOX did not metabolize any of the tested compounds (kcat < 0.05 s−1) that acted as non-competitive inhibitors having Ki ≥ 155 µM for SMOX. In addition, we tested (R,R)-1,12-bis-methylspermine (2,13-diamino-5,10-diazatetradecane) (R,R)-(Me2Spm) and (S,S)-Me2Spm as substrates for Fms1. Fms1 preferred (S,S)- to (R,R)-diastereoisomer, but with notably lower kcat 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.
Highlights
The polyamines spermidine (Spd) and spermine (Spm) and their diamine precursor putrescine (Put) are essential cellular constituents in eukaryotic organisms [1] (Figure 1A)
We have recently shown that polyamine transport system and the key enzymes of polyamine metabolism, namely ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AdoMetDC) and SSAT are divergently regulated by chiral C-methylated polyamine analogues [27,28]
We have shown that the stimulatory effect of aldehydes on the acetylpolyamine oxidase (APAO)-catalysed oxidation of the polyamines is based on the in situ formation of comparatively unstable Schiff base between the primary amino group of the polyamine and the aldehyde, i.e. an aldimine mimicking the charge distribution of N-acetylated polyamines (Figure 2) [29,32]
Summary
The polyamines spermidine (Spd) and spermine (Spm) and their diamine precursor putrescine (Put) are essential cellular constituents in eukaryotic organisms [1] (Figure 1A). Dysregulation of SMOX and activated Spm catabolism are associated with c 2018 The Author(s) We studied the substrate specificities of SMOX and APAO for N1-alkylated or N1-acylated derivatives of (R)- and (S)-MeSpd and the effects of supplemented aldehydes on Fms, that readily catalyses the oxidation of N1-acetylated Spd and Spm. We used (R,R)-Me2Spm and (S,S)-Me2Spm to gain insight into how 1,12-bis-methylation of Spm and configuration of chiral centres affects the substrate properties and binding to the active centre of Fms (Figure 1A). Obtained data demonstrate for the first time that stereospecificity and regiospecificity of FAD-dependent polyamine oxidases could be controlled with the conformationally restricted ligands exploiting existing conformational landscapes in enzyme without protein engineering
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
