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Updated: Mar 29, 2026
A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli
Published on: December 9, 2017
Regulation of a Protein Acetyltransferase in Myxococcus xanthus by the Coenzyme NADP
Xin-Xin Liu1, Wei-Bing Liu2, Bang-Ce Ye2
1Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Unlabelled:
NADP(+) is a vital cofactor involved in a wide variety of activities, such as redox potential and cell death. Here, we show that NADP(+) negatively regulates an acetyltransferase from Myxococcus xanthus, Mxan_3215 (MxKat), at physiologic concentrations. MxKat possesses an NAD(P)-binding domain fused to the Gcn5-type N-acetyltransferase (GNAT) domain. We used isothermal titration calorimetry (ITC) and a coupled enzyme assay to show that NADP(+) bound to MxKat and that the binding had strong effects on enzyme activity. The Gly11 residue of MxKat was confirmed to play an important role in NADP(+) binding using site-directed mutagenesis and circular dichroism spectrometry. In addition, using mass spectrometry, site-directed mutagenesis, and a coupling enzymatic assay, we demonstrated that MxKat acetylates acetyl coenzyme A (acetyl-CoA) synthetase (Mxan_2570) at Lys622 in response to changes in NADP(+) concentration. Collectively, our results uncovered a mechanism of protein acetyltransferase regulation by the coenzyme NADP(+) at physiological concentrations, suggesting a novel signaling pathway for the regulation of cellular protein acetylation.
Importance:
Microorganisms have developed various protein posttranslational modifications (PTMs), which enable cells to respond quickly to changes in the intracellular and extracellular milieus. This work provides the first biochemical characterization of a protein acetyltransferase (MxKat) that contains a fusion between a GNAT domain and NADP(+)-binding domain with Rossmann folds, and it demonstrates a novel signaling pathway for regulating cellular protein acetylation in M. xanthus. We found that NADP(+) specifically binds to the Rossmann fold of MxKat and negatively regulates its acetyltransferase activity. This finding provides novel insight for connecting cellular metabolic status (NADP(+) metabolism) with levels of protein acetylation, and it extends our understanding of the regulatory mechanisms underlying PTMs.
Insights
Nicotinamide adenine dinucleotide phosphate (NADP(+)) negatively regulates the Myxococcus xanthus acetyltransferase MxKat. This discovery reveals a new signaling pathway linking cellular metabolism to protein acetylation levels.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Microorganisms utilize protein posttranslational modifications (PTMs) for rapid environmental response.
- Protein acetylation is a crucial PTM, but its regulation is not fully understood.
Purpose of the Study:
- To biochemically characterize the Myxococcus xanthus acetyltransferase MxKat.
- To elucidate the regulatory mechanism of MxKat by NADP(+).
- To identify a novel signaling pathway for protein acetylation.
Main Methods:
- Isothermal titration calorimetry (ITC) and coupled enzyme assays.
- Site-directed mutagenesis and circular dichroism spectrometry.
- Mass spectrometry.
Main Results:
- NADP(+) binds to MxKat's NAD(P)-binding domain and negatively regulates its activity.
- The Gly11 residue is critical for NADP(+) binding.
- MxKat acetylates acetyl-CoA synthetase (Mxan_2570) at Lys622 in response to NADP(+) levels.
Conclusions:
- NADP(+) acts as a physiological regulator of MxKat activity.
- A novel signaling pathway connects NADP(+) metabolism to protein acetylation.
- This finding expands the understanding of PTM regulation in M. xanthus.
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