Intracellular acetyl phosphate modulates Escherichia coli pyruvate metabolism

Abstract

Lysine acetylation has been shown to be an abundant and vital post-translational modification (PTM) that utilizes acetyl phosphate (AcP) as one of the acetyl group donors in bacteria. The pyruvate dehydrogenase (PDH) complex catalyzes the conversion from pyruvate to acetyl coenzyme A (acetyl-CoA). Thus far, the connection between lysine acetylation and pyruvate metabolism has not been thoroughly investigated. In this study, we show that AcP could acetylate Escherichia coli pyruvate dehydrogenase (AceE) in vitro and in vivo, which could be reversed by protein lysine deacetylase (CobB). In vitro treatment of AceE with AcP also causes increased phosphorylation of the protein, whereas deleting ackA does not affect the phosphorylation of the protein. As a result, in vitro treatment of AceE by AcP leads to decreased enzymatic activity. In contrast, deleting ackA leads to increased acetylation and enzymatic activity of AceE, and deleting pta results in the decreased acetylation and enzymatic activity of AceE. As expected, deleting pta in E. coli causes pyruvate accumulation. Although deleting ackA also causes pyruvate accumulation, decreased expression of the two genes involved in pyruvate metabolism (ldhA and poxB) is observed in the mutant, indicating that AcP could affect pyruvate metabolism by other routes in addition to modulating the AceE activity. Thus, our results demonstrate that intracellular AcP could modulate pyruvate metabolism in E. coli. For the first time, a linkage between AcP-mediated protein lysine acetylation, pyruvate dehydrogenase activity, and pyruvate metabolism is established.

1. Introduction

Protein lysine acetylation is an abundant and vital post-translational modification across all domains of life that is involved in central metabolism, transcription regulation, protein synthesis, cell morphology, the cell cycle, signal pathway regulation, the stress response, and pathogenic microorganism infection regulation [1,2]. It occurs through two different routes. The first route is enzymatic, relying on lysine acetyltransferases (KATs), such as YfiQ (patZ), RimI, YiaC, YjaB, and PhnO [1], to transfer an acetyl group from acetyl-CoA to Nε-lysine residues [2]. The second route is non-enzymatic, in which acetyl phosphate donates its acetyl group to the ε-amino group of lysine residues [3‒5]. Protein lysine acetylation is reversible, and acetylated lysine can be enzymatically deacetylated by lysine deacetylases (KDACs). In Escherichia coli, the Sirtuin family protein CobB can deacetylate both enzymatically acetylated and non-enzymatically acetylated lysines [6].

Acetyl phosphate (AcP) was first found to be a precursor of acetic acid during fermentation, which is involved in acetate activation for its metabolic functions [7]. Phosphate acetyltransferase (Pta) catalyzes the conversion between acetyl-CoA and AcP, and acetate kinase (AckA) catalyzes the conversion between AcP and acetate. Both reactions are readily reversible. Cells lacking AckA can convert acetyl-CoA to AcP but cannot degrade AcP to acetate, leading to elevated AcP levels [8]. In contrast, cells lacking both Pta and AckA cannot synthesize AcP from either AcCoA or acetate, which completely abolishes AcP production. Previous studies showed that AcP could be a phosphoryl and an acetyl group donor for protein post-translational modifications (PTMs) [9]. In E. coli, acetylation of K243 by AcP inhibits the oriC binding ability of DNA [10]. In Salmonella, acetylation of K102 inhibits the activity of PhoP [11]. In Mycobacterium tuberculosis, AcP-dependent acetylation of the cyclic AMP receptor protein affects the virulence of the bacterium [12]. In Actinobacteria, AcP-dependent modification networks regulate c-di-AMP homeostasis [13]. In Baccilus subtili, AcP-mediated lysine acetylation modulates the activity of acetyl-CoA synthetase [14]. However, Bacillus subtilis DegU can be phosphorylated by AcP in the absence of DegS [15]. AcP has also been identified in vivo as an alternative source of CiaR phosphorylation in the absence of CiaH [16]. In Staphylococcus aureus, AcP-mediated phosphorylation inhibits the enzyme activity of SrtA [17]. However, the physiological roles of AcP-dependent acetylation or phosphorylation remain largely unknown.

Pyruvate is located at a significant metabolic node, which links carbohydrate catabolism to energy generation and biosynthesis and represents a major switch point between respiratory and fermentative metabolism [18]. The pyruvate dehydrogenase (PDH) complex converts pyruvate to acetyl coenzyme A (acetyl-CoA). The E. coli PDH complex has three components: pyruvate dehydrogenase (E1, AceE), dehydrolipoate acyltransferase (E2, AceF), and dihydrolipoate dehydrogenase (E3, LPD) [18]. AceE catalyzes the decarboxylation of pyruvate, a key step in the pathway.