Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation–π interaction

Abstract

Polyamines influence medically relevant processes in the opportunistic pathogen Pseudomonas aeruginosa, including virulence, biofilm formation and susceptibility to antibiotics. Although homospermidine synthase (HSS) is part of the polyamine metabolism in various strains of P. aeruginosa, neither its role nor its structure has been examined so far. The reaction mechanism of the nicotinamide adenine dinucleotide (NAD+)-dependent bacterial HSS has previously been characterized based on crystal structures of Blastochloris viridis HSS (BvHSS). This study presents the crystal structure of P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of the catalytically relevant residues is demonstrated, qualifying BvHSS as a model for mechanistic studies of PaHSS. Following this strategy, crystal structures of single-residue variants of BvHSS are presented together with activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues are required at the entrance to the binding pocket (`ionic slide') and near the active site (`inner amino site') to attract and bind the substrate putrescine via salt bridges. The tryptophan residue at the active site stabilizes cationic reaction components by cation–π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of this tryptophan for other amino acids suggests a distinct catalytic requirement for an aromatic interaction partner with a highly negative electrostatic potential. These findings substantiate the structural and mechanistic knowledge on bacterial HSS, a potential target for antibiotic design.

Rational engineering of homospermidine synthase for enhanced catalytic efficiency toward spermidine synthesis

“…2 B, Bv HSS residues E210 and D361 form hydrogen bonds with the N atom at homospermidine nitrogen 1 (inner amine site), while E237 forms a similar bond with the N atom at homospermidine nitrogen 11 (outer amine site). Additionally, N162 and W229 form hydrogen bonds and cation-π interactions with the N atom at homospermidine nitrogen 6 (center amine site) [ 18 ]. Spermidine shares a structural resemblance with homospermidine, featuring three amino groups and a carbon chain that is one methylene group shorter.…”

“…This Schiff base is subsequently reduced to an imino group, resulting in the production of homospermidine. The entire catalytic process relies on the coordination among residues Asn-162, Trp-229, Glu-237, His-296, and the nicotine amide ring of NAD + [ 17 , 18 ]. 1,3-diaminopropane and putrescine share similar structural and physicochemical characteristics.…”

Insights into the Unusual Activity of a Novel Homospermidine Synthase with a Promising Application to Produce Spermidine

“…35 So far, only some HSS from Blastochloris viridis (formerly known as Rhodopseudomonas viridis), 36 Pseudomonas aeruginosa, 37 Senecio vulgaris, 38 and Acinetobacter tartarogenes 39 have been characterized and identified, and the crystal structure and catalytic mechanism of HSS from Blastochloris viridis and Pseudomonas aeruginosa have been analyzed and reported in detail. 15,37 Although these studies discussed the substrate specificity of HSS, none of them advocate for employing HSS to produce spermidine. The ability of mutant BvHSS-D361E to catalyze the synthesis of spermidine was considerably superior to that of the wild-type, according to our previous research.…”

“…Homospermidine synthase is the first pathway-specific enzyme evolved from deoxyhypusine synthase (DHS, EC 2.5.1.46) in the biosynthesis of pyrrolizidine alkaloids . So far, only some HSS from Blastochloris viridis (formerly known as Rhodopseudomonas viridis), Pseudomonas aeruginosa, Senecio vulgaris, and Acinetobacter tartarogenes have been characterized and identified, and the crystal structure and catalytic mechanism of HSS from Blastochloris viridis and Pseudomonas aeruginosa have been analyzed and reported in detail. , Although these studies discussed the substrate specificity of HSS, none of them advocate for employing HSS to produce spermidine. The ability of mutant Bv HSS-D361E to catalyze the synthesis of spermidine was considerably superior to that of the wild-type, according to our previous research.…”

“…The primary distinction is that Pf HSS reacts with putrescine and 1,3-diaminopropane 7.2 times faster than Bv HSS. The catalytic reaction process of Pf HSS was speculated based on the results of the structure alignment and the resolution of the catalytic mechanism of Bv HSS by Scheidig and co-workers. , First, E236, H295, and E297 act as the catalytic triad, providing a strongly basic environment and attracting positively charged substrates to the active center. The hydroxide ions hydrolyze an amino group on the substrate via nucleophilic attack, removing ammonium ions into hydroxyl groups, and NAD + accepts a proton to form NADH.…”