Structural and catalytic characterization of B. viridis and P. aeruginosa homospermidine synthases reveals the essential role of cation-π interaction

Polyamines influence medically relevant processes in the opportunistic pathogen P. aeruginosa including virulence, biofilm formation and susceptibility to antibiotics. Although the pseudomonal homospermidine synthase (HSS) is part of the polyamine metabolism in various strains including PA7 and PA14, neither its role nor structure were examined so far. The reaction mechanism of the NAD+-dependent bacterial HSS was previously characterized based on crystal structures of the B. viridis HSS (BvHSS). This thesis presents the crystal structure of the P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of catalytically relevant residues is demonstrated, qualifying BvHSS as model for the reaction mechanism of PaHSS. Following this strategy, crystal structures of single residue variants of BvHSS are presented together with HPLC-based activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues at the entrance of the binding pocket (“ionic slide”) and near the active site (“inner amino site”) attract and coordinate putrescine, respectively. The tryptophan residue at the active site stabilizes cationic reaction components via cation-π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of the tryptophan residue for other amino acids revealed a distinct catalytic requirement for an aromatic interaction partner with highly negative electrostatic potential. The electron density distribution in one active site suggests formation of an NAD+ adduct. These findings deepen the structural and mechanistic knowledge about the bacterial HSS and provide approaches for rational inhibitor design.