Journal:Acta Cryst D:S2059798321008937
From Proteopedia
Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation-π interactionF. Helfrich and Axel J. Scheidig [1] Molecular Tour The highly conserved bacterial homospermidine synthase (HSS) is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila, Brucella spp., and various Pseudomonas aeruginosa strains[2]. The enzyme HSS is required for the NAD-dependent synthesis of the polyamine homospermidine (HSP) from the diamine putrescine (PUT)[3] (see static image below).  Recently we have determined the crystal structures of two bacterial HSS, HSS from Blastochloris viridis (BvHSS) and from Pseudomonas aeruginosa (PaHSS). BvHSS exists as a homo-dimeric enzyme in solution, whereas the PaHSS is monomeric in solution but displays the same as BvHSS[1],[4]. (PDB ID: 6y87). The domain 1 (“NAD(P)-binding Rossmann-like”) is colored in yellow (subunit A)/gold (subunit B) and domain 2 (“homospermidine synthase (HSS)-like”) in deep sky blue (subunit A)/royal blue (subunit B). The . The solvent-accessible surface of the binding pocket for subunit A is depicted in transparent red. The NAD+ molecule lining the surface of the pocket is shown as ball-and-stick representation. The cofactor NAD(H) is bound as a prosthetic group in the binding pocket with its nicotinamide ring being part of the active site. An “ionic slide” (BvHSS residues D94 and E117[4]) was proposed to lead positively charged amine substrates from the entrance of the binding pocket into the active site. The entrance tunnel is thereby lined by a so-called “track-and-trace” loop (BvHSS residues 114-130 [4]). Both enzymes display structural characteristics at their active site suggesting cation-π interaction through a highly conserved tryptophan as an important contribution for the catalyzed reaction. Geometry for cation-π interaction between the PUT atoms C4 and N2 and the tryptophan benzene moiety in BvHSS and PaHSS. BvHSS, PDB ID 4tvb, chain B[4]: PaHSS, PDB ID 6y87, chain E[1]:
Structures are given as ball-and-stick representation, distances as lines, angle legs as yellow lines for θ and white for φ; angle plane is in yellow for θ and in white for φ. The orthogonal projections of C4 and N2 onto the ring planes are shown as white spheres (C4’ and N2’). All angle legs originate from the centroid of the benzene moiety, including the distance vectors (centroid to C4 and N2). Angle θ is spanned by the normal of the ring plane and the C4 or N2 distance vector. Angle φ is between the vector pointing to C4’ or N2’ and the vector pointing to ring carbon CH2. Polyamines Polyamines are involved in various processes in nearly all organisms in the three domains of life[5]. In P. aeruginosa, polyamines and polyamine-related processes were demonstrated to be involved in growth[6], biofilm formation[7],[8],[9], susceptibility to antibiotics and exogenous polyamines[10],[11],[12],[13] as well as expression of the type III secretion system, a major virulence determinant[14],[15],[16]. Therefore, enzymes like HSS might be promising targets for new antibiotics. Proposed reaction mechanism of bacterial HSS  Based on crystal structures of the BvHSS, including the wildtype enzyme and several single-residue variants, a reaction mechanism depending on certain residues and the stably bound cofactor NAD(H) was proposed[4]. Acidic residues were suggested to attract and guide the substrate PUT via its positively charged amino groups into the binding pocket of the enzyme and to stabilize the substrate at the active site. The proposed reaction mechanism can be simplified and subdivided into two major parts as follows. First, one terminal carbon atom (atom C4) of PUT is oxidized by NAD+, forming NADH and an imine (step (1) to (3)). The imine is subsequently deaminated by nucleophilic attack of a water molecule, which yields a 4-aminobutanal (step (4)). The second part comprises another nucleophilic attack at atom C4 by the amino group of another PUT molecule (step (5/6)), yielding a Schiff base (step (7)). HSP is finally produced by electron transfer from NADH to the Schiff base, regenerating the oxidized NAD+ cofactor (step (8)). Based on the interaction geometry at the active site between the side chain of a conserved tryptophan residue and (I) the positively charged ammonium group as well as (II) the C4 atom of bound PUT and HSP molecules, cation-π interaction was suggested. In the course of the reaction, a positive charge is delocalized between carbon atom C4 and nitrogen atom N5. This charge is energetically stabilized by the π-electron system of the neighbouring indole ring of Trp229 (numbering based on BvHSS). A geometric analysis of the positioning of the indole ring and the substrates suggests that the formation of a positive partial charge on C4 is energetically favoured and therefore stabilized. Overall, the indole ring of residue BvHSS-Trp229 (correspondingly PaHSS-Trp225) is proposed to be involved in the stabilization of transient carbocations and positively charged nitrogen atoms (e.g. protonated imine (step 3) and the protonated Schiff base (step 7)) via cation-π interactions, thus playing a major role in lowering the transition state energy, intermediate stabilization and conversion of reaction components. Significantly reduced activity could only be retained by replacement of the tryptophan residue by the aromatic residues phenylalanine or tyrosine, supporting the requirement for an aromatic system as cation-π-interaction partner. PDB references: homospermidine synthase from Blastochloris viridis, W229E variant, complex with NAD, 6sep; E210Q variant, complex with NAD, 6s3x; W229F variant, complex with NAD, 6s4d; E210A variant, complex with NAD, 6s49; E117Q variant, complex with NAD, 6s6g; W229A variant, complex with NAD and PUT, 6s72; from Pseudomonas aeruginosa, complex with NAD and PUT, 6y87. References
| |||||||||||
