Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum

Abstract

Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of l-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction was characterized. It was shown that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting the hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions.

Effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor, OdhI, involved in glutamate production inCorynebacterium glutamicum

“…6) It is likely that in the L-glutamate-producing conditions, a large portion of the carbon flux is shifted toward the anaplerotic pathway mediated by phosphoenolpyruvate carboxylase, rather than the acetyl phosphate and acetate-producing Pta-AckA pathways, with the resulting depletion of acetylation substrates. 6,7) In this condition, succinylation utilizing succinyl-CoA would readily occur. Although we have not identified an enzyme responsible for K132 succinylation, a non-enzymatic mechanism might be considered, as suggested in mitochondria.…”

“…6) We also showed that phosphoenolpyruvate carboxylase, which is an anaplerotic enzyme catalyzing the supply of oxaloacetate and which is essential for L-glutamate production, is regulated by protein acetylation. 7) However, the functions of acylation of most other proteins remain to be elucidated.…”

Post-translational Protein Acetylation: An Elegant Mechanism for Bacteria to Dynamically Regulate Metabolic Functions

“…It is now clear that the extent of acetylation, the mechanisms by which acetylation occurs, the identities of acetylated proteins, and the locations of acetylation on those proteins can vary between organisms; yet, in most cases, there is great conservation in core cellular processes (Nakayasu et al, 2017). Such approaches have shown that acetylation occurs on proteins involved in a wide diversity of cellular process, especially central metabolism and translation (Wang et al, 2010; Weinert et al, 2013a; Baeza et al, 2014; Kuhn et al, 2014; Kosono et al, 2015; Schilling et al, 2015; Kentache et al, 2016; Birhanu et al, 2017; Jers et al, 2017; Nagano-Shoji et al, 2017; Post et al, 2017; Bontemps-Gallo et al, 2018; Gaviard et al, 2018; Sun et al, 2018; Vasileva et al, 2018; Yoshida et al, 2019). As stated above, many of these acetylations are conserved.…”

Improvement of Xylose Utilization and L-ornithine Production by Metabolic Engineering of Corynebacterium glutamicum

“…Phosphoenolpyruvate and carbon dioxide synthesize oxaloacetic acid under the catalysis of PEPC, and then oxaloacetic acid enters the TCA cycle for further metabolism. The expression and activity of PEPC affects the synthesis of glutamic acid in C. glutamicum [46,47] . Lysine at position 653 (K653) is essential for the regulation of PEPC acetylation.…”

“…found that acetylation of PEPC at K653 could decrease enzymatic activity and glutamate production, which refer to the K653-acetylation could regulate PEPC activity negatively. Mutated K653 into arginine could decrease the level of acetylation on PEPC, which correspondingly improve its activity [47] . In addition, PEPC would be feedback suppressed by aspartic acid when it was overexpressed in C. glutamicum.…”