Journal:Acta Cryst D:S2059798321008937
From Proteopedia
(Difference between revisions)
| Line 6: | Line 6: | ||
Bacterial Homospermidine Synthase | 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<ref name="Shaw">PMID:20194510</ref>. The enzyme HSS is required for the NAD-dependent synthesis of the polyamine homospermidine (HSP) from the diamine putrescine (PUT) (Figure 1)<ref name="Tait">PMID:437275</ref>. 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<ref name="Krossa">PMID:26776105</ref>,<ref name="Helfrich1">PMID:34605434</ref>. | + | 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<ref name="Shaw">PMID:20194510</ref>. The enzyme HSS is required for the NAD-dependent synthesis of the polyamine homospermidine (HSP) from the diamine putrescine (PUT) (Figure 1)<ref name="Tait">PMID:437275</ref>. 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<ref name="Krossa">PMID:26776105</ref>,<ref name="Helfrich1">PMID:34605434</ref>. |
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<ref name="Krossa">PMID:26776105</ref>) 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 <ref name="Krossa">PMID:26776105</ref>). 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. | 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<ref name="Krossa">PMID:26776105</ref>) 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 <ref name="Krossa">PMID:26776105</ref>). 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 | ||
| - | Polyamines are involved in various processes in nearly all organisms in the three domains of life<ref name="Michael">PMID:27268252</ref>. In P. aeruginosa, polyamines and polyamine-related processes were demonstrated to be involved in growth<ref name="Bitonti">PMID:6818954</ref>, biofilm formation<ref name="Cardile">PMID:27864804</ref>,<ref name="Qu">PMID:26817804</ref> | + | Polyamines are involved in various processes in nearly all organisms in the three domains of life<ref name="Michael">PMID:27268252</ref>. In P. aeruginosa, polyamines and polyamine-related processes were demonstrated to be involved in growth<ref name="Bitonti">PMID:6818954</ref>, biofilm formation<ref name="Cardile">PMID:27864804</ref>,<ref name="Qu">PMID:26817804</ref>,<ref name="Williams">PMID:20149107</ref>, 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 | 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<ref name="Krossa">PMID:26776105</ref>. 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 | 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<ref name="Krossa">PMID:26776105</ref>. 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 | ||
| Line 28: | Line 28: | ||
| - | + | ||
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. | 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. | Zhou, L., Wang, J. & Zhang, L.-H. (2007). PloS one 2, e1291. | ||
Revision as of 12:05, 17 November 2021
| |||||||||||
This page complements a publication in scientific journals and is one of the Proteopedia's Interactive 3D Complement pages. For aditional details please see I3DC.
