Journal:Acta Cryst D:S2059798321008937

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

F. Helfrich and Axel J. Scheidig ^[1]

Molecular Tour
Bacterial Homospermidine Synthase

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) (Figure 1)^[3]. 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 dimeric arrangement in the crystal as BvHSS^[4],^[1].

The HSS is composed of two domains, an “NAD(P)-binding Rossmann-like domain” and an “HSS-like domain” (Figure 2). The substrate binding pocket is located between these two domains. 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. Polyamines Polyamines are involved in various processes in nearly all organisms in the three domains of life (Michael, 2016). In P. aeruginosa, polyamines and polyamine-related processes were demonstrated to be involved in growth (Bitonti et al., 1982), biofilm formation (Cardile et al., 2017; Qu et al., 2016; Williams et al., 2010), susceptibility to antibiotics and exogenous polyamines (Kwon & Lu, 2007; Kwon & Lu, 2006b; Kwon & Lu, 2006a, Yao, 2012 #346) as well as expression of the type III secretion system, a major virulence determinant (Anantharajah et al., 2016; Wu et al., 2012; Zhou et al., 2007). 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. Figure 1. Main reaction catalyzed by HSS. Two putrescine (PUT) molecules are converted into one sym-homospermidine (HSP) molecule. Figure 2. Cartoon representation of the dimeric PaHSS as observed in the crystal structure (PDB ID: 6Y87). The domain 1 (“NAD(P)-binding Rossmann-like”) is colored in yellow (subunit A)/orange (subunit B) and domain 2 (“homospermidine synthase (HSS)-like”) in blue (subunit A)/dark blue (subunit B). The solvent-accessible surface of the binding pocket for subunit A is depicted in transparent red with the entrance pointing upwards. The NAD+ molecule lining the surface of the pocket is shown as ball-and-stick representation. Figure 3. Proposed reaction steps of the conversion of PUT to HSP by the bacterial HSS. Relevant residues, NAD(H), PUT, HSP and intermediates are shown as two-dimensional structure representations. Hydrogen bonds are depicted as blue dotted lines, delocalized electrons as dashed lines, cation-π interactions as orange dash-dotted lines and electron transfers as red arrows. Atom numbering is given for PUT and HSP in green. For simplicity, steps 5 and 6 are shown in combined depictions with correspondingly labeled electron transfers. A more detailed sequence of reaction steps was described before^[4] and additional intervening reaction steps are proposed in Fig. S2 of Helfrich & Scheidig, 2021^[1]. Figure 4. Geometry of cation-π interaction between the PUT atoms C4 and N2 and the tryptophan benzene moiety in BvHSS (panel A and B, PDB ID 4TVB chain B^[4]) and PaHSS (panel C and D, PDB ID 6Y87 chain E^[1]). Structures are given as ball-and-stick representation, distances as yellow dashed lines, angle legs as grey lines and angles as grey transparent triangles (not visible for φ in (A)). The orthogonal projections of C4 and N2 onto the ring planes are shown as black spheres (C4’ and N2’). All angle legs originate from the centroid of the benzene moiety, including the dashed distance vectors (centroid to C4 and N2). Angle θ is spanned by the normal of the ring plane (grey, infinitely pointing upwards) and the C4 or N2 distance vector (yellow, dashed). Angle φ is between the vector pointing to C4’ or N2’ and the vector pointing to ring carbon CH2. References Anantharajah, A., Mingeot-Leclercq, M.-P. & van Bambeke, F. (2016). Trends Pharmacol. Sci. 37, 734–749. Bitonti, A. J., McCann, P. P. & Sjoerdsma, A. (1982). Biochem. J. 208, 435–441. Cardile, A. P., Woodbury, R. L., Sanchez, C. J., Becerra, S. C., Garcia, R. A., Mende, K., Wenke, J. C. & Akers, K. S. (2017). Adv. Exp. Med. Biol. 973, 53–70.

Kwon, D.-H. & Lu, C.-D. (2007). Antimicrob. Agents Chemother. 51, 2070–2077. Kwon, D. H. & Lu, C.-D. (2006a). Antimicrob. Agents Chemother. 50, 1615–1622. Kwon, D. H. & Lu, C.-D. (2006b). Antimicrob. Agents Chemother. 50, 1623–1627. Michael, A. J. (2016). J. Biol. Chem. 291, 14896–14903. Qu, L., She, P., Wang, Y., Liu, F., Zhang, D., Chen, L., Luo, Z., Xu, H., Qi, Y. & Wu, Y. (2016). Microbiologyopen 5, 402–412.

Tait, G. H. (1979). Biochem. Soc. Trans. 7, 199–201.

Williams, B. J., Du, R.-H., Calcutt, M. W., Abdolrasulnia, R., Christman, B. W. & Blackwell, T. S. (2010). Mol. Microbiol. 76, 104–119. Wu, D., Lim, S. C., Dong, Y., Wu, J., Tao, F., Zhou, L., Zhang, L.-H. & Song, H. (2012). J. Mol. Biol. 416, 697–712. Zhou, L., Wang, J. & Zhang, L.-H. (2007). PloS one 2, e1291.

References

1. ↑ ^{1.0 1.1 1.2 1.3} Helfrich F, Scheidig AJ. Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation-pi interaction. Acta Crystallogr D Struct Biol. 2021 Oct 1;77(Pt 10):1317-1335. doi:, 10.1107/S2059798321008937. Epub 2021 Sep 23. PMID:34605434 doi:http://dx.doi.org/10.1107/S2059798321008937
2. ↑ Shaw FL, Elliott KA, Kinch LN, Fuell C, Phillips MA, Michael AJ. Evolution and multifarious horizontal transfer of an alternative biosynthetic pathway for the alternative polyamine sym-homospermidine. J Biol Chem. 2010 May 7;285(19):14711-23. doi: 10.1074/jbc.M110.107219. Epub 2010, Mar 1. PMID:20194510 doi:http://dx.doi.org/10.1074/jbc.M110.107219
3. ↑ Tait GH. The formation of homospermidine by an enzyme from Rhodopseudomonas viridis [proceedings]. Biochem Soc Trans. 1979 Feb;7(1):199-201. doi: 10.1042/bst0070199. PMID:437275 doi:http://dx.doi.org/10.1042/bst0070199
4. ↑ ^{4.0 4.1 4.2 4.3 4.4 4.5} Krossa S, Faust A, Ober D, Scheidig AJ. Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase-an Essential Enzyme of the Polyamine Metabolism. Sci Rep. 2016 Jan 18;6:19501. doi: 10.1038/srep19501. PMID:26776105 doi:http://dx.doi.org/10.1038/srep19501