Structural and Kinetic Characterization of the SpeG Spermidine/Spermine N-acetyltransferase from Methicillin-Resistant Staphylococcus aureus USA300

Abstract

Polyamines are simple yet critical molecules with diverse roles in numerous pathogenic and non-pathogenic organisms. Regulating polyamine concentrations affects the transcription and translation of genes and proteins important for cell growth, stress, and toxicity. One way polyamine concentrations are maintained within the cell is via spermidine/spermine N-acetyltransferases (SSATs) that acetylate intracellular polyamines so they can be exported. The bacterial SpeG enzyme is an SSAT that exhibits a unique dodecameric structure and allosteric site compared to other SSATs that have been previously characterized. While its overall 3D structure is conserved, its presence and role in different bacterial pathogens are inconsistent. For example, not all bacteria have speG encoded in their genomes; in some bacteria, the speG gene is present but has become silenced, and in other bacteria, it has been acquired on mobile genetic elements. The latter is the case for methicillin-resistant Staphylococcus aureus (MRSA) USA300, where it appears to aid pathogenesis. To gain a greater understanding of the structure/function relationship of SpeG from the MRSA USA300 strain (SaSpeG), we determined its X-ray crystal structure in the presence and absence of spermine. Additionally, we showed the oligomeric state of SaSpeG is dynamic, and its homogeneity is affected by polyamines and AcCoA. Enzyme kinetic assays showed that pre-incubation with polyamines significantly affected the positive cooperativity toward spermine and spermidine and the catalytic efficiency of the enzyme. Furthermore, we showed bacterial SpeG enzymes do not have equivalent capabilities to acetylate aminopropyl versus aminbutyl ends of spermidine. Overall, this study provides new insight that will assist in understanding the SpeG enzyme and its role in pathogenic and non-pathogenic bacteria at a molecular level.

Keywords: Gcn5-related N-acetyltransferase (GNAT); MRSA USA300; Staphylococcus aureus; acetylation; polyamine; spermidine/spermine N-acetyltransferase (SSAT).

Figure 1

Kinetic characterization of SaSpeG using two different assays. The difference between Assay 1 and 2 was that the SaSpeG enzyme was not pre-incubated with polyamine prior to Assay 1 compared to pre-incubation in Assay 2. See Materials and Methods for additional assay details. (A) Assay 1 results of polyamine substrate saturation curves. Data were fitted with both Michaelis–Menten (MM) and an allosteric sigmoidal equation in Prism 8.0. The MM equation fitting is indicated with a black line, whereas fitting with allosteric sigmoidal is in pink for spermine and purple for spermidine. Log plots (second row) are shown to clarify the fitting between the two equations. (B) Assay 2 results of polyamine substrate saturation curves. Similar to panel A, data were fitted to two equations (MM and allosteric sigmoidal), and log plots are shown. The MM equation fitting is indicated with a red line, whereas fitting with the allosteric sigmoidal equation is shown in blue for spermine and black for spermidine. (C) Comparison of Assay 1 and Assay 2 results after normalization for both polyamines. Curves are colored as mentioned in panels A and B. (D) Substrate saturation curves for AcCoA in the presence of spermine (spm) and spermidine (spd) as described in Materials and Methods.

Figure 2

Products of enzymatic spermidine acetylation by SpeG enzymes. (A) UV chromatograms (293 nm) of the acetylated products of a 5 min reaction (solid gray line) of SaSpeG overlaid with the injections of 200 µM standards of N¹-acetylspermidine (black dashed line) and N⁸-acetylspermidine (blue dashed line). (B) Extracted ion chromatogram of the reaction products corresponding to a proton adduct of the dansylated acetylspermidine (m/z 654). (C) UV chromatograms (293 nm) of the acetylated products for VcSpeG (solid orange line) compared to standards as in panel A. (D) UV chromatograms (293 nm) of the acetylated products for all tested SpeGs. SaSpeG (gray), VcSpeG (orange), EcSpeG (green), and BtSpeG (purple). Data for BtSpeG is the same as previously reported in [29]. Ratios of N8-acetylspermidine to N1-acetylspermidine products were SaSpeG (1:1.13), VcSpeG (1:6.23), EcSpeG (1:0.97) and BtSpeG (1:1.02).

Figure 3

Structure of SaSpeG. (A) SaSpeG protomer displays an α/β GNAT fold. (B) Topological representation of SaSpeG protomer consists of 4 α-helices and 7-β strands with GNAT motifs A–D highlighted. (C) SaSpeG dodecamer is a functional unit of the enzyme formed by a dimer of hexamers. Two GNAT dimers, chain A and chain B, form one GNAT dimer, and chain C and chain D form the other. Interface-1 interactions lie within GNAT dimer, chain A and chain B or chain C and chain D, Interface-2 interactions between chain B and chain D or chain A and chain C, Interface-3 interactions are present between chain A and chain D or chain B and chain C. (D) Two SaSpeG protomers of the same hexamer with bound spermine molecule in the allosteric site.

Figure 4

Comparison of SaSpeG and VcSpeG acceptor and allosteric sites within a single monomer. (A) SaSpeG protein (PDB ID 8fv1 chain B) with AcCoA modeled from the VcSpeG PDB ID 4r57 structure. The donor site contains AcCoA in magenta, the allosteric site in complex with spermine is shown in green, and the V-splay of the acceptor site is highlighted in gold. (B) SaSpeG PDB ID 8fv1 chain B structure with allosteric and acceptor site residues highlighted as described in panel A. The conserved catalytic tyrosine residue Y129 is shown in grey. (C) VcSpeG PDB ID 4r57 chain A structure with allosteric site residues in purple and active site residues in magenta, and critical catalytic Y134 residue is in grey.

Figure 5

SaSpeG structure in complex with spermine and representative Gram-positive and Gram-negative SpeG protein sequence alignment. (A) Structural alignment of the SaSpeG apo form (gray, PDB ID 5IX3) and SaSpeG in complex with spermine (spm) (yellow and light green PDB ID 8FV0 and 8FV1, respectively) demonstrating the flexibility of the loop between helices α1 and α2 to accommodate the binding of spermine in the allosteric site. Loop movement is indicated with a red arrow. (B) Residues E29, E33, H45, D48, E49, E51 form interactions with spermine. (C) Surface of the dodecamer with twelve spermine molecules positioned within the allosteric sites of each monomer within the dodecamer. (D) Multiple sequence alignment using MultAlin [33] and then visualized using ESPript 3.0 [34]. Protein sequences include Staphylococcus aureus USA300 (SaSpeG), Bacillus thuringenisis (BtSpeG), Yersinia pestis (YpSpeG), Escherichia coli (EcSpeG), Salmonella typhimurium SL1344 (StSpeG), and Vibrio cholerae (VcSpeG) from UniProt IDs: A0A0H2XGJ0, A0A437SL45, A0A5P8YIA2, P0A951, A0A719HIP0, and Q9KL03, respectively. The secondary structure of the SaSpeG protein crystal structure (PDB ID 8FV0) is shown above the alignment. Conserved amino acids are highlighted in red.

Figure 6

Evaluation of SaSpeG oligomerization using Native PAGE, EMSA, and SEC. (A,B) Native PAGE of SaSpeG at different concentrations of protein and in presence or absence of different concentrations of spermine (spm) and/or AcCoA. Protein was purified from cells not grown in auto-induction media. (C) EMSA of SaSpeG in presence and absence of spm, spermidine (spd), and/or AcCoA. Protein was purified from cells grown in auto-induction media. (D) Size-exclusion chromatograms of the SaSpeG protein in presence and absence of spermine (spm) produced four elution peakes labeled 1–4, respectively.

Figure 7

Electrostatic and hydrophobic surfaces of Gram-positive and Gram-negative bacterial SpeG crystal structures. Dodecamer electrostatic surfaces (top two rows) are shown in red (negative), blue (positive), and white (neutral). Distribution of hydrophobic and aromatic residues (alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan) on dodecamers are shown in orange (middle two rows). Distribution of aromatic residues (phenylalanine, tryptophan, tyrosine) in green compared to small hydrophobic residues (alanine, valine, isoleucine, leucine) in orange (bottom two rows). The full surface of the protein is shown in gray. Top and side (rotated 90º along y-axis) views of each dodecamer are shown for each representation. PDB IDs are listed beneath columns of structures; apo structures are shown with gray bars, and liganded structures with purple bars. All liganded structures were in complex with spermine, with the exception of 3wr7, which was in complex with both spermine and CoA.