SpeG polyamine acetyltransferase enzyme from Bacillus thuringiensis forms a dodecameric structure and exhibits high catalytic efficiency

Abstract

Polyamines are important for regulating biofilms and the exopolysaccharide of the biofilm matrix of Bacillus subtilis. Understanding how enzymes can regulate polyamine concentrations is critical for learning more about how these processes occur in diverse bacteria. Here, we describe the structure and function of another member of the spermidine/spermine acetyltransferases (SSAT) found in Bacilli. The SpeG enzyme from B. thuringiensis (BtSpeG) binds polyamines in its allosteric site and adopts a dodecameric oligomeric state similar to other SpeG enzymes from Gram-negative bacteria. Our kinetic results show the catalytic efficiency of BtSpeG was greater than any previously characterized SpeG to date, and in contrast to other SpeG proteins it exhibited very similar kinetic properties toward both spermine and spermidine. Similar to the SpeG enzyme from E. coli, BtSpeG was able to acetylate spermidine on the N¹ and N⁸ positions. The turnover of BtSpeG toward spermine and spermidine was also two to three orders of magnitude greater than any other Bacilli SSAT enzyme that has been previously characterized. SpeG proteins from Bacilli, including B. cereus, B. thuringiensis and B. anthracis share nearly identical sequences and therefore our results likely provide insight into the structure/function relationship across multiple Bacillus species.

Keywords: Bacillus; Dodecamer; Gcn5-related NAcetyltransferase; Polyamines; SpeG; X-ray crystallography.

Figure 1.

A, Diamond shaped SpeG crystals in ligand-free form were obtained in Tris pH 7.5, PEG 4000, and 0.2 M lithium sulfate. B, SpeG crystals grown in the presence of spermine and 30% ethanol produced small irregular crystals. Small single crystals could be mounted in the loop. C, Tertiary structure of BtSpeG monomer in cartoon format. Secondary structures including β-strands, α-helices, and connecting loops colored in purple, cyan and orange respectively. The BtSpeG protomer is comprised of a 7-stranded anti-parallel sheet, with the exception of parallel strands β5 and β6, wrapped around 4-α -helices. D, The secondary structure elements represent the N-acetyltransferase sequence motifs C (β1-α1), D (β2—β3), A (β4-α3) and B (β5-α4) highlighted in colored boxes in blue, green, red and brown, respectively. The least conserved secondary structure elements Motif C, β-strand 6 and β7 and helix α2, can be absent in some GNAT proteins. Topology of BtSpeG and associated primary sequence is presented in cartoon form. E, A dimer was present in the asymmetric unit of BtSpeG in ligand-free form. A spermine molecule positioned in the allosteric site is shown in stick mode (red), and modelled AcCoA molecule (yellow) is shown in the active site (yellow).

Figure 2.. Dodecamer of BtSpeG produces three binding interfaces.

A, Biological unit of BtSpeG is composed of a dimer of hexamers with D6 symmetry. Larger letters correspond to the monomers on the top face of the hexamer and the smaller letters correspond to monomers on the bottom face of the hexamer. Three interfaces mediate assembly of the dodecameric biological unit with two hexamers stacked on top of each other with D6 symmetry. B, schematic representation of dodecameric assembly of BtSpeG. C, Schematic representation of the four monomers that produce the 3 binding interfaces involved in biological unit assembly with schematic representation. The type I interface is between neighboring monomers of the same hexamer (A/B) and (A/ F). The type II interface is formed between monomers (A/ G) of opposing hexamers. The type III interface is formed between diagonal adjacent partners of the opposing hexamers.

Figure 3.. Spermine interactions with BtSpeG.

A, Schematic representation of detailed interaction between spermine and BtSpeG dimer. Hydrogen bonds are represented by dotted lines. B, graphical representation of bond formed between spermine and two monomers of BtSpeG molecule, monomer A in green and B in cyan. Spermine binds in the allosteric site between two monomers that form a type I interface of the structure. C, Superimposition of flexible loop region residues 25–38 of BtSpeG in ligand-bound (green) and ligand-free (magenta) state. Movement of the flexible loop is necessary in order to accommodate the spermine molecule in the allosteric site to avoid a clash. D, Dodecameric assembly of BtSpeG biological unit (cyan) with twelve spermine molecules in the allosteric site (purple) and putative active site with twelve AcCoA molecules (yellow and green).

Figure 4.. Structural alignment of BtSpeG (6VFN), VcSPeG (4R87) and VcSpeG (6E1X).

A, Structural alignment of GNAT dimer of BtSpeG (6VFN) in light cyan, VcSpeG (4R87) in light green and VcSpeG (6E1X) in light magenta revealed no significant structural differences. B, Enlarged view of active site alignment with visible acetylated spermine (ac-seprmine) in red from VcSpeG (6E1X) shows no significant differences. C, Zoomed in view of allosteric site with spermine molecule (red) of three SpeG protomers shows nearly identical configuration. D, Close view of highly conserved active site with a CoA molecule (orange) shows no significant structural differences.

Figure 5.. Kinetic characterization of BtSpeG.

A, Reaction time course at RT, no preincubation of enzyme with spermine. BtSpeG enzyme concentration was 150 nM and the reaction contained 70 mM Bicine pH 7.8, 20 mM NaCl, 4 mM spermine, and 1 mM AcCoA. B, Reaction time course at RT, with preincubation of enzyme with spermine. BtSpeG enzyme concentration was 17.5 nM. C, Activity of BtSpeG pre-incubated with spermine as a function of pH of Bicine buffer. Each reaction contained 70 mM Bicine buffer at different pH values, 20 mM NaCl, 4 mM spermine, and 1 mM AcCoA and reactions were performed for 5 min at 37°C. Enzyme was preincubated with spermine. D, Substrate saturation curves of BtSpeG toward spermine (black triangles) and spermidine (red circles). Enzyme was preincubated with either spermine or spermidine depending upon which polyamine was being assayed. 4 nM enzyme was used for spermine saturation curves and 8.5 nM enzyme was used for spermidine saturation curves. All reactions were performed in duplicate and the average and standard deviation of at least two biological replicates are shown. E, Identification of products of enzymatic acetylation of spermidine by LC/MS. Products of the 5 min reaction containing 11.8 nM BtSpeG, 4 mM spermidine, and 1 mM AcCoA at 22 deg C (Bt SpeG +spd) are compared to 200 uM solutions of N1 and N8 acetylspermidine standards. The bottom trace is the ion chromatogram for the fully dansylated acetylspermidine extracted using a 20 ppm window.