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. 2024 Jun;300(6):107404.
doi: 10.1016/j.jbc.2024.107404. Epub 2024 May 21.

Structure and dynamics of the staphylococcal pyridoxal 5-phosphate synthase complex reveal transient interactions at the enzyme interface

Affiliations

Structure and dynamics of the staphylococcal pyridoxal 5-phosphate synthase complex reveal transient interactions at the enzyme interface

Angélica Luana C Barra et al. J Biol Chem. 2024 Jun.

Abstract

Infectious diseases are a significant cause of death, and recent studies estimate that common bacterial infectious diseases were responsible for 13.6% of all global deaths in 2019. Among the most significant bacterial pathogens is Staphylococcus aureus, accounting for more than 1.1 million deaths worldwide in 2019. Vitamin biosynthesis has been proposed as a promising target for antibacterial therapy. Here, we investigated the biochemical, structural, and dynamic properties of the enzyme complex responsible for vitamin B6 (pyridoxal 5-phosphate, PLP) biosynthesis in S. aureus, which comprises enzymes SaPdx1 and SaPdx2. The crystal structure of the 24-mer complex of SaPdx1-SaPdx2 enzymes indicated that the S. aureus PLP synthase complex forms a highly dynamic assembly with transient interaction between the enzymes. Solution scattering data indicated that SaPdx2 typically binds to SaPdx1 at a substoichiometric ratio. We propose a structure-based view of the PLP synthesis mechanism initiated with the assembly of SaPLP synthase complex that proceeds in a highly dynamic interaction between Pdx1 and Pdx2. This interface interaction can be further explored as a potentially druggable site for the design of new antibiotics.

Keywords: PLP synthase; Staphylococcus aureus; oligomeric state; protein crystallization; vitamin B6.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
PLP synthesis by SaPdx1 and SaPLP synthase.A, the Schiff base concentration over time is increased for SaPLP synthase (SaPdx1 + SaPdx2, cyan curve) compared to SaPdx1 and ammonia (orange curve). The linear fit of the concentration over time shows an increase of 2.5 times favoring SaPLP synthase. B and C, michaelis‒Menten kinetics as a function of R5P (B) and G3P (C). The obtained kinetic parameters are shown in Table 1. These results were obtained from three independent experiments, each performed in triplicate. G3P, glyceraldehyde 3-phosphate; PLP, pyridoxal 5-phosphate; R5P, ribose 5-phosphate.
Figure 2
Figure 2
SAXS data of SaPdx1 obtained in batch (buffer 1) or SEC-SAXS (buffers 2 and 3) measurements. Different buffer conditions are colored in yellow (buffer 1: 50 mM Tris–HCl pH 8, 150 mM NaCl), purple (buffer 2: 50 mM Tris–HCl pH 8, 200 mM Na2SO4), and green (buffer 3: 100 mM Na2HPO4 pH 8, 150 mM NaCl). A, solution scattering X-ray intensity pattern in relative intensity units with their respective Guinier plots (inset) (B) p(r) functions in relative scale. C, dimensionless Kratky plots with the globular protein reference as the dotted line. Ab initio GASBOR models in orthogonal views, their fits (χ2) with the experimental data, and superimposed with the crystallographic structure of SaPdx1 are shown in cartoon representation (D) in buffer 1, (E) buffer 2, and (F) buffer 3. SAXS, small-angle X-ray scattering; SEC, size-exclusion chromatography.
Figure 3
Figure 3
PLP synthesis in different buffers.A, PLP synthesis by SaPdx1 in the presence of R5P, G3P, and ammonia. The activity measured in buffer 1 (50 mM Tris–HCl pH 8, 150 mM NaCl) was lower than that in buffer 2 (50 mM Tris–HCl pH 8, 200 mM Na2SO4) or buffer 3 (100 mM Na2HPO4 pH 8, 150 mM NaCl). The inset shows that the initial delay in enzyme activity is also increased when buffer 1 is used. These results were obtained from three independent experiments, each performed in triplicate. B, SEC-MALS analysis of SaPdx1 in different buffers. Prior to substrate addition, SaPdx1 was found to be dodecameric in buffers 2 and 3 and in equilibrium of hexamers and dodecamers in buffer 1, favoring hexamers. After adding the substrates R5P, G3P, and ammonia, the enzyme was found to be a dodecamer in all buffers evaluated. These results were obtained from two independent experiments. G3P, glyceraldehyde 3-phosphate; MALS, multiangle light scattering; PLP, pyridoxal 5-phosphate; R5P, ribose 5-phosphate; SEC, size-exclusion chromatography.
Figure 4
Figure 4
Crystal structure of SaPdx1.A, the asymmetric unit content has two SaPdx1 monomers. B, a closer view of the SaPdx1 monomer: the distorted 8 strand is shown in yellow. An EDO molecule is shown in the active site interacting with lysine K82. The catalytic lysine K150 and the phosphate bound to the P2 site are indicated. C and D, two orthogonal views of the SaPdx1 dodecamer assembled by the H 32 crystal symmetry.
Figure 5
Figure 5
SaPLP assembly.A, size-exclusion chromatography of the PLP complex in buffer 4 (50 mM Tris–HCl, pH 8, 150 mM NaCl, 10 mM L-glutamine). In cyan lines, the SEC profile for the SaPdx1-2wt. SDS‒PAGE (inset) shows that peaks I and II contain SaPdx1-2wt, while peak III contains free Pdx2wt. The purple line shows the SEC profile for SaPdx1-2mut with a single peak containing the SaPdx1–SaPdx2mut complex. The gel lane labeled as Ap shows the sample prior to SEC analysis, while the sample labeled as I shows SaPdx1–SaPdx2mut complex. These results were obtained from five independent experiments. B, SEC-MALS analysis confirmed unsaturated SaPdx1-2wt species, as well as some free SaPdx2 without mutation (cyan line). For SaPdx1-2mut (purple line), the equilibrium is shifted toward the fully saturated PLP complex. After adding the substrates (cyan dashed line), SaPdx1-2wt is also shifted toward a fully saturated complex. The results were obtained from two independent experiments. PLP, pyridoxal 5-phosphate; SAXS, small-angle X-ray scattering.
Figure 6
Figure 6
SAXS analysis of the SaPLP synthase complex obtained in SEC-SAXS measurements.Left: scattering data (dots) and best fit of the SaPdx1-2wt sample composition (cyan line), as estimated by OLIGOMER. The composition is shown in the inset. Right: scattering data (dots) and best fit of the SaPdx1-2mut composition (purple line), as estimated by OLIGOMER. The composition is shown in the inset. SaPLP, Staphylococcus aureus pyridoxal 5-phosphate; SAXS, small-angle X-ray scattering; SEC, size-exclusion chromatography.
Figure 7
Figure 7
Crystal structure of the SaPLP complex.A, overall arrangement of the complex. SaPdx1 is shown colored by secondary structure, while SaPdx2mut is shown as a yellow cartoon. B, a glutamine amino acid (gray sticks) is bound to the SaPdx2mut active site, shown in yellow. C, crystal structure of the SaPLP complex colored by crystallographic B factors. A color key is shown in Å2 units. D, a detailed view of the interactions between SaPdx1 and SaPdx2mut in the SaPLP complex. SaPLP, Staphylococcus aureus pyridoxal 5-phosphate.

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