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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.

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.

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).

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