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Structure and activity of the DHNA Coenzyme-A Thioesterase from Staphylococcus aureus providing insights for innovative drug development - PubMed

  • ️Sat Jan 01 2022

Structure and activity of the DHNA Coenzyme-A Thioesterase from Staphylococcus aureus providing insights for innovative drug development

Aline Melro Murad et al. Sci Rep. 2022.

Abstract

Humanity is facing an increasing health threat caused by a variety of multidrug resistant bacteria. Within this scenario, Staphylococcus aureus, in particular methicillin resistant S. aureus (MRSA), is responsible for a number of hospital-acquired bacterial infections. The emergence of microbial antibiotic resistance urgently requires the identification of new and innovative strategies to treat antibiotic resistant microorganisms. In this context, structure and function analysis of potential drug targets in metabolic pathways vital for bacteria endurance, such as the vitamin K2 synthesis pathway, becomes interesting. We have solved and refined the crystal structure of the S. aureus DHNA thioesterase (SaDHNA), a key enzyme in the vitamin K2 pathway. The crystallographic structure in combination with small angle X-ray solution scattering data revealed a functional tetramer of SaDHNA. Complementary activity assays of SaDHNA indicated a preference for hydrolysing long acyl chains. Site-directed mutagenesis of SaDHNA confirmed the functional importance of Asp16 and Glu31 for thioesterase activity and substrate binding at the putative active site, respectively. Docking studies were performed and rational designed peptides were synthesized and tested for SaDHNA inhibition activity. The high-resolution structure of SaDHNA and complementary information about substrate binding will support future drug discovery and design investigations to inhibit the vitamin K2 synthesis pathway.

© 2022. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

(a) Cartoon representation of the SaDHNA monomer. (b) The secondary structure of SaDHNA is shown schematically and annotated including the topology plot of its HotDog domain. (c) The quaternary arrangement of SaDHNA is shown for the individual monomer chains with chain A in blue, B in yellow, C in red and D in green. The figure was created applying the program PyMOL (Molecular Graphics System, Version 1.0.5.4 Schrödinger, LLC).

Figure 2
Figure 2

DLS and SAXS data of SaDHNA. (a) Autocorrelation function and corresponding mean radius distribution obtained by DLS, averaged over 300 s. (b) Averaged X-ray scattering intensities of SaDHNA (blue dots) in arbitrary units plotted against the scattering angle, i.e. momentum transfer values ranging from s = 0.05 to 2.4 nm−1. The red fit function corresponds to the SaDHNA ab initio model displayed in figure panel C, sharing a χ2-value of 1.54 with the corresponding experimental scattering data. (c) Superimposition of a single SaDHNA ab initio model calculated applying the program GASBOR and the tetramer of SaDHNA revealed by the crystal structure.

Figure 3
Figure 3

(a) Superimposition of SaDHNA (dark blue) with homologous structures, PDB codes 1Z54 of Thermus. thermophylus (pink) and 1BVQ of Pseudomonas sp (cyan). (b) Protein sequence alignment. The light blue and red boxes illustrate the highest variability compared between the homologue enzymes. Multiple sequence alignment was performed applying the program ClustalOmega with default parameters.

Figure 4
Figure 4

Putative active sites of the tetrameric SaDHNA-CoA thioesterase. (a) Ribbon representation of SaDHNA (light pink) superimposed with the 4-hydroxybenzoyl-CoA thioesterase D17N mutant structure of Pseudomonas (PDB code 1LO9) (light grey). The substrate 4-hydroxybenzoyl-CoA (BCA) complexed with the D17N Ps4HBT mutant is shown in stick representation; carbon in cyan; sulfur in yellow; phosphate in orange, nitrogen in blue, oxygen in red; “N” and “C” indicate N- and C-terminus, respectively. (b) Residues at the proposed active site region with assigned thioesterase activity of SaDHNA (pink), Pseudomonas 4HBT structure (yellow) and hypothetical thioesterase from Thermus. thermophilus (pdb code 1Z54) (light gray). Italic amino acid residue labels indicate corresponding residues in the Pseudomonas 4HBT structure (yellow).

Figure 5
Figure 5

Fluorescence emission spectra of SaDHNA (60 nM) in the absence and the presence of the gradual concentrations of (a) Pep-1 and (b) Pep-2 up to 1000 nM.

Figure 6
Figure 6

(a) Stern–Volmer plot for Pep-1 and Pep-2 ranging from 0 to 1000 nM. (b) Stern–Volmer plot indicating the bimolecular quenching constant (Kq). (c) Static quenching plot to estimate the affinity-binding constant (Ka).

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