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Lysine acetylation of Escherichia coli lactate dehydrogenase regulates enzyme activity and lactate synthesis.

ABSTRACT: As an evolutionarily conserved posttranslational modification, protein lysine acetylation plays important roles in many physiological and metabolic processes. However, there are few reports about the applications of lysine acetylation in metabolic regulations. Lactate is a main byproduct in microbial fermentation, and itself also an important bulk chemical with considerable commercial values in many fields. Lactate dehydrogenase (LdhA) is the key enzyme catalyzing lactate synthesis from pyruvate. Here, we reported that Escherichia coli LdhA can be acetylated and the acetylated lysine sites were identified by mass spectrometry. The effects and regulatory mechanisms of acetylated sites on LdhA activity were characterized. Finally, lysine acetylation was successfully used to regulate the lactate synthesis. LdhA (K9R) mutant overexpressed strain improved the lactate titer and glucose conversion efficiency by 1.74 folds than that of wild-type LdhA overexpressed strain. LdhA (K154Q-K248Q) mutant can inhibit lactate accumulation and improve 3HP production. Our study established a paradigm for lysine acetylation in lactate synthesis regulation and suggested that lysine acetylation may be a promising strategy to improve the target production and conversion efficiency in microbial synthesis. The application of lysine acetylation in regulating lactate synthesis also provides a reference for the treatment of lactate-related diseases.

SUBMITTER: Liu M

PROVIDER: S-EPMC9424733 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Lysine acetylation of <i>Escherichia coli</i> lactate dehydrogenase regulates enzyme activity and lactate synthesis.

Liu Min M Huo Meitong M Liu Changshui C Guo Likun L Ding Yamei Y Ma Qingjun Q Qi Qingsheng Q Xian Mo M Zhao Guang G

Frontiers in bioengineering and biotechnology 20220816

As an evolutionarily conserved posttranslational modification, protein lysine acetylation plays important roles in many physiological and metabolic processes. However, there are few reports about the applications of lysine acetylation in metabolic regulations. Lactate is a main byproduct in microbial fermentation, and itself also an important bulk chemical with considerable commercial values in many fields. Lactate dehydrogenase (LdhA) is the key enzyme catalyzing lactate synthesis from pyruvate ...[more]

PMID: 36051589

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Project description:Nε-lysine acetylation is a common posttranslational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription factors, little is known about how this posttranslational modification regulates protein activity. In this work, we investigated how lysine acetylation affects translation in Escherichia coli. In multiple species of bacteria, ribosomal proteins are highly acetylated at conserved lysine residues, suggesting that this modification may regulate translation. In support of this hypothesis, we found that the addition of either of the acetyl donors acetyl phosphate and acetyl-coenzyme A inhibits translation but not transcription using an E. coli cell-free system. Further investigations using in vivo assays revealed that acetylation does not appear to alter the rate of translation elongation but, rather, increases the proportions of dissociated 30S and 50S ribosomes, based on polysome profiles of mutants or growth conditions known to promote lysine acetylation. Furthermore, ribosomal proteins are more acetylated in the disassociated 30S and 50S ribosomal subunits than in the fully assembled 70S complex. The effect of acetylation is also growth rate dependent, with disassociation of the subunits being most pronounced during late-exponential and early-stationary-phase growth-the same growth phase where protein acetylation is greatest. Collectively, our data demonstrate that lysine acetylation inhibits translation, most likely by interfering with subunit association. These results have also uncovered a new mechanism for coupling translation to the metabolic state of the cell. IMPORTANCE Numerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from central metabolite acetyl-coenzyme A or acetyl phosphate to a lysine residue in a protein. This posttranslational modification is known to regulate some central metabolic enzymes and transcription factors in bacteria, though a comprehensive understanding of its effect on cellular physiology is still lacking. In the present study, lysine acetylation was also found to inhibit translation in Escherichia coli by impeding ribosome association, most likely by disrupting salt bridges along the binding interface of the 30S and 50S ribosomal subunits. These results further our understanding of lysine acetylation by uncovering protein synthesis as a new target of regulation and aid in the design of bacteria for biotechnology applications where the growth conditions are known to promote lysine acetylation.

Project description:Antibiotic resistance is increasingly becoming a challenge to public health. The regulation of bacterial metabolism by post-translational modifications (PTMs) has been widely studied. However, the mechanism underlying the regulation of acetylation in bacterial resistance to antibiotics is still unknown. Here, we performed a quantitative analysis of the acetylated proteome of a wild-type (WT) Escherichia coli (E. coli) sensitive strain and ampicillin- (Re-Amp), kanamycin- (Re-Kan), and polymyxin B-resistant (Re-Pol) strains. Based on bioinformatics analysis combined with biochemical validations, we found a common regulatory mechanism between the different resistant strains. Our results showed that protein acetylation negatively regulates bacterial metabolism to regulate antibiotic resistance and positively regulates bacterial motility. Further analyses revealed that key enzymes in various metabolic pathways were differentially acetylated. In particular, pyruvate kinase (PykF), a glycolytic enzyme that regulates bacterial metabolism, and its acetylated form were highly expressed in the three resistant strains and were identified as reversibly acetylated by the deacetylase CobB and the acetyl-transferase PatZ (peptidyl-lysine N-acetyltransferase). Results showed that PykF also could be acetylated by nonenzymatic acetyl phosphatase (AcP) in vitro. Furthermore, the deacetylation of Lys413 in PykF increased PykF enzymatic activity by changing the conformation of its ATP binding site, resulting in an increase in energy production which, in turn, increased the sensitivity of drug-resistant strains to antibiotics. This study provides novel insights for understanding bacterial resistance and lays the foundation for future research on the regulation of acetylation in antibiotic-resistant strains. IMPORTANCE The misuse of antibiotics has resulted in the emergence of many antibiotic-resistant strains which seriously threaten human health. Protein post-translational modifications, especially acetylation, tightly control bacterial metabolism. However, the comprehensive mechanism underlying the regulation of acetylation in bacterial resistance remains unexplored. Here, acetylation was found to positively regulate bacterial motility and negatively regulate energy metabolism, which was common in all antibiotic-resistant strains. Moreover, the acetylation and deacetylation process of PykF was uncovered, and deacetylation of the Lys 413 in PykF was found to contribute to bacterial sensitivity to antibiotics. This study provides a new direction for research on the development of bacterial resistance through post-translational modifications and a theoretical basis for developing antibacterial drugs.

Dataset Information

Lysine acetylation of Escherichia coli lactate dehydrogenase regulates enzyme activity and lactate synthesis.

Publications

Lysine acetylation of <i>Escherichia coli</i> lactate dehydrogenase regulates enzyme activity and lactate synthesis.

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