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.
The Spermine/Spermidine N‐acetyltransferase from Escherichia coli (EcSpeG) is an allosteric dodecameric enzyme that belongs to the Gcn5‐related N‐acetyltransferase (GNAT) superfamily of enzymes. Under stressful conditions, SpeG acetylates the polyamines spermine (spm) and spermidine (spd), which bind to the active site and allosteric site of the protein. These polyamines are important polycationic molecules for many life processes including cell growth, protein synthesis, and metabolic regulation. GNATs commonly use a general acid base catalytic mechanism to acetylate their substrates and most have a key tyrosine residue that acts as the general acid in the reaction. It was previously shown that mutating this conserved residue in EcSpeG to phenylalanine did not abrogate the catalytic activity. In our experience, mutating this conserved residue in other GNATs has shown it is absolutely critical for activity. Therefore, we created multiple point mutations to test whether the identity of the residue at this position altered catalytic activity. Similar to our previous results with other GNATs, we found this tyrosine residue was critical for activity. Support or Funding Information Research reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).
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.
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