Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Baumgartner, Jackson; Mohammad, Thahani S. Habeeb; Czub, M.P.; Majorek, K.A.; Arolli, Xhulio; Variot, Cillian; Anonick, Madison; Minor, W.; Ballícora, Miguel A.; Becker, Daniel P.; Kuhn, Misty L.

doi:10.3389/fmolb.2021.646046

Cited by 9 publications

(23 citation statements)

References 77 publications

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“…We recently showed a serine residue in a comparable position to EcSpeG Y135 in a GNAT that does not acetylate polyamines is critical for enzymatic activity and uses a different chemical mechanism. 5 Therefore, we created this point mutation and tested the mutant enzyme for activity; it exhibited the most significant decrease in activity of all of the mutants we tested. Thus, substituting a variety of amino acids at this position, even if they are critical for activity in other GNATs, did not yield a catalytically robust EcSpeG enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…[1][2][3] Most GNATs that have been characterized use acetyl coenzyme A (AcCoA) as a donor substrate to acetylate broad acceptor substrates ranging in size from small molecules to larger proteins. They tend to accomplish this via a general acid/base chemical mechanism, but other mechanisms have been observed [reviewed in 4,5 ]. In a general acid/base mechanism, one of the residues in the active site acts as a general base to abstract a proton from the acceptor substrate so the acceptor can perform a nucleophilic attack on the carbonyl carbon of the donor substrate.…”

Section: Introductionmentioning

confidence: 99%

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

Dang

Lim

et al. 2021

Protein Science

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Dang

Lim

et al. 2021

Protein Science

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“…Recently, a novel catalytic mechanism was reported for the P. aeruginosa PA3944 GNAT, which shows activity toward polymyxin antibiotics (Baumgartner et al, 2021). This catalytic mechanism involves a catalytic serine residue directly acting as nucleophile resulting in the formation of a covalent acylenzyme intermediate (Figure 2C).…”

Section: An Active Site Tyrosine That Might Act As General Acid Is Essential For Catalysismentioning

confidence: 98%

“…This catalytic mechanism involves a catalytic serine residue directly acting as nucleophile resulting in the formation of a covalent acylenzyme intermediate (Figure 2C). It was shown that the glutamate originally regarded as catalytic base plays a role in substrate recognition or stabilization (Baumgartner et al, 2021). If this mechanism also applies to certain GNAT protein acetyltransferases needs further investigation.…”

Section: An Active Site Tyrosine That Might Act As General Acid Is Essential For Catalysismentioning

confidence: 99%

Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective

Lammers

2021

Front. Microbiol.

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Ac(et)ylation is a post-translational modification present in all domains of life. First identified in mammals in histones to regulate RNA synthesis, today it is known that is regulates fundamental cellular processes also in bacteria: transcription, translation, metabolism, cell motility. Ac(et)ylation can occur at the ε-amino group of lysine side chains or at the α-amino group of a protein. Furthermore small molecules such as polyamines and antibiotics can be acetylated and deacetylated enzymatically at amino groups. While much research focused on N-(ε)-ac(et)ylation of lysine side chains, much less is known about the occurrence, the regulation and the physiological roles on N-(α)-ac(et)ylation of protein amino termini in bacteria. Lysine ac(et)ylation was shown to affect protein function by various mechanisms ranging from quenching of the positive charge, increasing the lysine side chains’ size affecting the protein surface complementarity, increasing the hydrophobicity and by interfering with other post-translational modifications. While N-(ε)-lysine ac(et)ylation was shown to be reversible, dynamically regulated by lysine acetyltransferases and lysine deacetylases, for N-(α)-ac(et)ylation only N-terminal acetyltransferases were identified and so far no deacetylases were discovered neither in bacteria nor in mammals. To this end, N-terminal ac(et)ylation is regarded as being irreversible. Besides enzymatic ac(et)ylation, recent data showed that ac(et)ylation of lysine side chains and of the proteins N-termini can also occur non-enzymatically by the high-energy molecules acetyl-coenzyme A and acetyl-phosphate. Acetyl-phosphate is supposed to be the key molecule that drives non-enzymatic ac(et)ylation in bacteria. Non-enzymatic ac(et)ylation can occur site-specifically with both, the protein primary sequence and the three dimensional structure affecting its efficiency. Ac(et)ylation is tightly controlled by the cellular metabolic state as acetyltransferases use ac(et)yl-CoA as donor molecule for the ac(et)ylation and sirtuin deacetylases use NAD+ as co-substrate for the deac(et)ylation. Moreover, the accumulation of ac(et)yl-CoA and acetyl-phosphate is dependent on the cellular metabolic state. This constitutes a feedback control mechanism as activities of many metabolic enzymes were shown to be regulated by lysine ac(et)ylation. Our knowledge on lysine ac(et)ylation significantly increased in the last decade predominantly due to the huge methodological advances that were made in fields such as mass-spectrometry, structural biology and synthetic biology. This also includes the identification of additional acylations occurring on lysine side chains with supposedly different regulatory potential. This review highlights recent advances in the research field. Our knowledge on enzymatic regulation of lysine ac(et)ylation will be summarized with a special focus on structural and mechanistic characterization of the enzymes, the mechanisms underlying non-enzymatic/chemical ac(et)ylation are explained, recent technological progress in the field are presented and selected examples highlighting the important physiological roles of lysine ac(et)ylation are summarized.

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“…In general, GNATs utilize either a direct acyl transfer mechanism where an acyl donor directly reacts with an acceptor substrate or a ping-pong mechanism involving an acyl-enzyme intermediate. 27 The GNAT domain of TblD exhibits moderate similarity Scheme 2. Chemical Synthesis of 11 to the glutamate N α -acetyltransferase PolN in the biosynthesis of polyoxins, 28 which reportedly uses a catalytic serine residue (Ser22) to form an acetyl-enzyme intermediate during catalysis.…”

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confidence: 99%

Characterization of Enzymes Catalyzing the Initial Steps of the β-Lactam Tabtoxin Biosynthesis

2022

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Tabtoxin is a β-lactam ring-containing phytotoxin produced by a plant pathogenic Pseudomonas species. Here, we describe the early stages of tabtoxin biosynthesis, involving a C-methylation reaction catalyzed by the S-adenosyl-l-methionine-dependent methyltransferase TblA as the initial step for the β-lactam construction. Gene deletion and in vitro biochemical assays demonstrated that the Gcn5-related N-acetyltransferase domain of TblD catalyzes the acetylation of the α-amino group of 5-methyl-l-lysine. This establishment of the early reaction steps lays the foundation for characterizing unique β-lactam biosynthesis.

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Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Cited by 9 publications

References 77 publications

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

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

Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective

Characterization of Enzymes Catalyzing the Initial Steps of the β-Lactam Tabtoxin Biosynthesis

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