Gcn5-related N-acetyltransferases (GNATs) are found in all kingdoms of life and catalyze important acyl transfer reactions in diverse cellular processes. While many 3D structures of GNATs have been determined, most do not contain acceptor substrates in their active sites. To expand upon existing crystallographic strategies for improving acceptor-bound GNAT structures, we synthesized peptide substrate analogs and reacted them with CoA in PA4794 protein crystals. We found two separate mechanisms for bisubstrate formation: 1) a novel X-ray induced radical-mediated alkylation of CoA with an alkene peptide, and 2) direct alkylation of CoA with a halogenated peptide. Our approach is widely applicable across the GNAT superfamily and can be used to improve the success rate of obtaining liganded structures of other acyltransferases.
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.
28Spermidine N-acetyltransferase (SpeG) acetylates and thus neutralizes toxic 29 polyamines. Studies indicate that SpeG plays an important role in virulence and 30 pathogenicity of many bacteria, which have evolved SpeG-dependent strategies 31 to control polyamine concentrations and survive in their hosts. In Escherichia coli, 32 the two-component response regulator RcsB is reported to be subject to N ε -33 acetylation on several lysine residues, resulting in reduced DNA binding affinity 34 and reduced transcription of the small RNA rprA; however, the physiological 35 acetylation mechanism responsible for this behavior has not been fully determined. 36Here, we performed an acetyltransferase screen and found that SpeG inhibits rprA 37 promoter activity in an acetylation-independent manner. Surface plasmon 38 resonance analysis revealed that SpeG can physically interact with the DNA-39 binding carboxyl domain of RcsB. We hypothesize that SpeG interacts with the 40 DNA-binding domain of RcsB and that this interaction might be responsible for 41SpeG-dependent inhibition of RcsB-dependent rprA transcription. This work 42 provides a model for SpeG as a modulator of E. coli transcription through its ability 43 to interact with the transcription factor RcsB. This is the first study to provide 44 evidence that an enzyme involved in polyamine metabolism can influence the 45 function of the global regulator RcsB, which integrates information concerning 46 envelope stresses and central metabolic status to regulate diverse behaviors. 47
Spermidine N-acetyltransferase (SpeG) acetylates and thus neutralizes toxic polyamines. Studies indicate that SpeG plays an important role in virulence and pathogenicity of many bacteria, which have evolved SpeG-dependent strategies to control polyamine concentrations and survive in their hosts. In Escherichia coli, the two-component response regulator RcsB is reported to be subject to Nε-acetylation on several lysine residues, resulting in reduced DNA binding affinity and reduced transcription of the small RNA rprA; however, the physiological acetylation mechanism responsible for this behavior has not been fully determined. Here, we performed an acetyltransferase screen and found that SpeG inhibits rprA promoter activity in an acetylation-independent manner. Surface plasmon resonance analysis revealed that SpeG can physically interact with the DNA-binding carboxyl domain of RcsB. We hypothesize that SpeG interacts with the DNA-binding domain of RcsB and that this interaction might be responsible for SpeG-dependent inhibition of RcsB-dependent rprA transcription. This work provides a model for SpeG as a modulator of E. coli transcription through its ability to interact with the transcription factor RcsB. This is the first study to provide evidence that an enzyme involved in polyamine metabolism can influence the function of the global regulator RcsB, which integrates information concerning envelope stresses and central metabolic status to regulate diverse behaviors.
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