Journal:Acta Cryst D:S2059798321008937
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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>,<ref name="Williams">PMID:20149107</ref>, susceptibility to antibiotics and exogenous 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>,<ref name="Williams">PMID:20149107</ref>, susceptibility to antibiotics and exogenous polyamines<ref name="Kwon">PMID:17438056</ref>,<ref name="Kwonb">PMID:16641427</ref>,<ref name="Kwona">PMID:16641426</ref> ( 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 | ||
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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<ref name="Krossa">PMID:26776105</ref> and additional intervening reaction steps are proposed in Fig. S2 of Helfrich & Scheidig, 2021<ref name="Helfrich1">PMID:34605434</ref>. | 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<ref name="Krossa">PMID:26776105</ref> and additional intervening reaction steps are proposed in Fig. S2 of Helfrich & Scheidig, 2021<ref name="Helfrich1">PMID:34605434</ref>. | ||
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<ref name="Krossa">PMID:26776105</ref>) and PaHSS (panel C and D, PDB ID 6Y87 chain E<ref name="Helfrich1">PMID:34605434</ref>). 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. | 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<ref name="Krossa">PMID:26776105</ref>) and PaHSS (panel C and D, PDB ID 6Y87 chain E<ref name="Helfrich1">PMID:34605434</ref>). 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. | ||
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Anantharajah, A., Mingeot-Leclercq, M.-P. & van Bambeke, F. (2016). Trends Pharmacol. Sci. 37, 734–749. | Anantharajah, A., Mingeot-Leclercq, M.-P. & van Bambeke, F. (2016). Trends Pharmacol. Sci. 37, 734–749. | ||
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| - | 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. | ||
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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 12:11, 17 November 2021
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