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

Tsimbalyuk, S.; Shornikov, Aleksander; Le, Van Thi Bich; Kuhn, Misty L.; Forwood, Jade K.

doi:10.1016/j.jsb.2020.107506

Cited by 6 publications

(13 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Proteins were expressed and purified using procedures previously described in (Tsimbalyuk et al, 2020). All crystallization trials were performed using the hanging drop vapor diffusion method in 48-well plates (Hampton Research).…”

Section: Crystallizationmentioning

confidence: 99%

“…Catabolic enzymes that are critical for regulating polyamine concentrations include the spermidine/spermine N-acetyltransferases (SSATs). These enzymes acetylate polyamines using acetyl coenzyme A (AcCoA) as an acetyl donor (Zhu et al, 2006;Hegde et al, 2007;Filippova et al, 2015a;Tsimbalyuk et al, 2020). Once acetylated, the overall charge of the polyamine is reduced and the molecules are exported (Kramer et al, 2008) or recycled (Floris and Finazzi Agrò, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…Once acetylated, the overall charge of the polyamine is reduced and the molecules are exported (Kramer et al, 2008) or recycled (Floris and Finazzi Agrò, 2013). Several different SSATs have been investigated, including hSSAT1 (Homo sapiens) (Zhu et al, 2006), PaiA (Thermoplasma acidophilum, Bacillus subtillis) (Forouhar et al, 2005;Filippova et al, 2011), BltD (Bacillus subtilis) (Woolridge et al, 1999), and SpeG (Vibrio cholerae, E. coli, Coxiella burnetti, Staphylococcus aureus, and Bacillus thuringiensis) (Fukuchi et al, 1994;Filippova et al, 2015a;Franklin et al, 2015;Li et al, 2019;Tsimbalyuk et al, 2020). All SSATs that have been kinetically characterized have been shown to acetylate spm more readily or at least as well as other polyamines.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The Vibrio cholerae SpeG Spermidine/Spermine N-Acetyltransferase Allosteric Loop and β6-β7 Structural Elements Are Critical for Kinetic Activity

Tsimbalyuk

Lim

et al. 2021

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

Polyamines regulate many important biological processes including gene expression, intracellular signaling, and biofilm formation. Their intracellular concentrations are tightly regulated by polyamine transport systems and biosynthetic and catabolic pathways. Spermidine/spermine N-acetyltransferases (SSATs) are catabolic enzymes that acetylate polyamines and are critical for maintaining intracellular polyamine homeostasis. These enzymes belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily and adopt a highly conserved fold found across all kingdoms of life. SpeG is an SSAT protein found in a variety of bacteria, including the human pathogen Vibrio cholerae. This protein adopts a dodecameric structure and contains an allosteric site, making it unique compared to other SSATs. Currently, we have a limited understanding of the critical structural components of this protein that are required for its allosteric behavior. Therefore, we explored the importance of two key regions of the SpeG protein on its kinetic activity. To achieve this, we created various constructs of the V. cholerae SpeG protein, including point mutations, a deletion, and chimeras with residues from the structurally distinct and non-allosteric human SSAT protein. We measured enzyme kinetic activity toward spermine for ten constructs and crystallized six of them. Ultimately, we identified specific portions of the allosteric loop and the β6-β7 structural elements that were critical for enzyme kinetic activity. These results provide a framework for further study of the structure/function relationship of SpeG enzymes from other organisms and clues toward the structural evolution of members of the GNAT family across domains of life.

show abstract

Section: Crystallizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The Vibrio cholerae SpeG Spermidine/Spermine N-Acetyltransferase Allosteric Loop and β6-β7 Structural Elements Are Critical for Kinetic Activity

Tsimbalyuk

Lim

et al. 2021

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

show abstract

“…For example, the SSAT1 protein from human and mouse are all homodimers with domain swapping between protomers, 6,9 but PaiA proteins are monomers 10 and SpeG proteins can be dodecamers that do not exhibit domain swapping. [11][12][13][14] The active sites of all of these proteins have a conserved tyrosine residue that has been suggested to act as a general acid in the human and mouse SSAT1 proteins 6,15 (Figure 1(a)). However, the role of this residue in the bacterial SpeG protein has been brought into question by Sugiyama et al 14 When this conserved tyrosine residue was mutated to phenylalanine…”

Section: Introductionmentioning

confidence: 99%

“…Although all SSAT proteins adopt a GNAT fold, their oligomeric states differ significantly. For example, the SSAT1 protein from human and mouse are all homodimers with domain swapping between protomers, 6,9 but PaiA proteins are monomers 10 and SpeG proteins can be dodecamers that do not exhibit domain swapping 11–14 . The active sites of all of these proteins have a conserved tyrosine residue that has been suggested to act as a general acid in the human and mouse SSAT1 proteins 6,15 (Figure 1(a)).…”

Section: Introductionmentioning

confidence: 99%

Criticality of a conserved tyrosine residue in the SpeG protein from Escherichia coli

Dang

Lim

et al. 2021

Protein Science

Self Cite

View full text Add to dashboard Cite

The SpeG spermidine/spermine N-acetyltransferase (SSAT) from Escherichia coli belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins. In vitro characterization of this enzyme shows it acetylates the polyamines spermine and spermidine, with a preference toward spermine. This enzyme has a conserved tyrosine residue (Y135) that is found in all SSAT proteins and many GNAT functional subfamilies. It is located near acetyl coenzyme A in the active center of these proteins and has been suggested to act as a general acid in a general acid/base chemical mechanism. In contrast, a previous study showed this residue was not critical for E. coli SpeG enzymatic activity when mutated to phenylalanine. This result was quite different from previous studies with a comparable residue in the human and mouse SSAT proteins, which also acetylate spermine and spermidine. Therefore, we constructed several mutants of the E. coli SpeG Y135 residue and tested their enzymatic activity. We found this conserved residue was indeed critical for E. coli SpeG enzyme activity and may behave similarly in other SSAT proteins.

show abstract

A polyamine acetyltransferase regulates the motility and biofilm formation of Acinetobacter baumannii

et al. 2023

View full text Add to dashboard Cite

Acinetobacter baumannii is a nosocomial pathogen highly resistant to environmental changes and antimicrobial treatments. Regulation of cellular motility and biofilm formation is important for its virulence, although it is poorly described at the molecular level. It has been previously reported that Acinetobacter genus specifically produces a small positively charged metabolite, polyamine 1,3-diaminopropane, that has been associated with cell motility and virulence. Here we show that A. baumannii encodes novel acetyltransferase, Dpa, that acetylates 1,3-diaminopropane, directly affecting the bacterium motility. Expression of dpa increases in bacteria that form pellicle and adhere to eukaryotic cells as compared to planktonic bacterial cells, suggesting that cell motility is linked to the pool of non-modified 1,3-diaminopropane. Indeed, deletion of dpa hinders biofilm formation and increases twitching motion confirming the impact of balancing the levels of 1,3-diaminopropane on cell motility. The crystal structure of Dpa reveals topological and functional differences from other bacterial polyamine acetyltransferases, adopting a β-swapped quaternary arrangement similar to that of eukaryotic polyamine acetyltransferases with a central size exclusion channel that sieves through the cellular polyamine pool. The structure of catalytically impaired DpaY128F in complex with the reaction product shows that binding and orientation of the polyamine substrates are conserved between different polyamine-acetyltransferases.

show abstract

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

Cited by 6 publications

References 52 publications

The Vibrio cholerae SpeG Spermidine/Spermine N-Acetyltransferase Allosteric Loop and β6-β7 Structural Elements Are Critical for Kinetic Activity

The Vibrio cholerae SpeG Spermidine/Spermine N-Acetyltransferase Allosteric Loop and β6-β7 Structural Elements Are Critical for Kinetic Activity

Criticality of a conserved tyrosine residue in the SpeG protein from Escherichia coli

A polyamine acetyltransferase regulates the motility and biofilm formation of Acinetobacter baumannii

Contact Info

Product

Resources

About