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Identification of EcpK, a bacterial tyrosine pseudokinase important for exopolysaccharide biosynthesis in Myxococcus xanthus

ABSTRACT: Bacteria secrete chemically diverse polysaccharides that function in many physiological processes and are widely used in industrial applications. In the ubiquitous Wzx/Wzy-dependent pathway for polysaccharide biosynthesis in Gram-negative bacteria, a polysaccharide co-polymerase (PCP) functions at the nexus between repeat-unit polymerization in the periplasm and polysaccharide translocation across the outer membrane by stimulating both processes. These PCPs are integral inner membrane proteins with extended periplasmic domains, and functionally depend on alternating between different oligomeric states. The oligomeric state, in turn, is determined by a cognate cytoplasmic bacterial Tyr kinase (BYK), which is either part of the PCP or a stand-alone protein, together with a Tyr phosphatase. Jointly, the BYK/phosphatase pair enables Tyr phosphorylation/dephosphorylation cycles, thereby facilitating the alternation between PCP oligomeric states. Interestingly, non-canonical BYK proteins, which lack key catalytic residues and/or the phosphorylated Tyr residues, have been described. In Myxococcus xanthus, the secreted polysaccharide EPS is synthesized and secreted via the Wzx/Wzy-dependent EPS pathway in which EpsV serves as the PCP. Here, we confirm that EpsV lacks the BYK domain. Using phylogenomics, experiments and computational structural biology, we identify EcpK as important for EPS biosynthesis and show that it structurally resembles canonical BYKs but lacks residues important for catalysis and Tyr phosphorylation. Using proteomic analyses, two-hybrid assays and structural modeling, we demonstrate that EcpK directly interacts with EpsV. Based on these findings, we suggest that EcpK functions as a scaffold and by direct protein-protein interactions, rather than by Tyr phosphorylation, facilitates EpsV activity. EcpK and EpsV orthologs are present in other bacteria, suggesting a broad conservation of this mechanism.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Myxococcus Xanthus

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD058227 | Pride | 2025-03-05

REPOSITORIES: pride

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Project description:Metabolism is fundamental to life, all life activities are sustained through metabolism. Metabolic engineering, while modifying host endogenous metabolic network or introducing heterologous pathway for biotechnological applications, often results in unfitness and suboptimal characteristics, because of the lack of full knowledge on host metabolism. Adaptive laboratory evolution (ALE), harnessing the nature of life -- evolution, has become a potent approach in phenotype optimization, environmental adaptation, and cell biology study1–3. Through ALE, alternative pathways of β-alanine biosynthesis4, pyridoxal 5’-phosphate (PLP) biosynthesis5, isoleucine biosynthesis6, as well as ATP generation7 have been uncovered; E. coli aerobic citrate utilization8,9 and various synthetic C1-trophy10–14 have been realized. In this study, we describe E. coli recruits the same workforce, Sad, in evolution through various strategies, i.e., catalytic efficiency improvement, gene dose increase, and gene expression regulations, to overcome metabolic damages (Fig. 1). Sad, i.e. NAD(P)+-dependent succinate semialdehyde dehydrogenase (SSADH), is an aldehyde dehydrogenase (ALDH) family protein. Sad oxidizes succinate semialdehyde to succinate, preventing accumulation of the toxic aldehyde intermediate in nitrogen metabolism, such as putrescine, arginine and γ-aminobutyrate (GABA) degradations15–17. Some bacteria, for example E. coli, possess also a NADP+-dependent SSADH, GabD15,18. SSADH is ubiquitous in both prokaryotes and eukaryotes. In human, it involves in catabolism of GABA, a major neurotransmitter, its malfunction leads to a disorder SSADH deficiency19. In plants, SSADH involves in leaf development and morphogenesis20 as well as defense of abiotic stress21. And in cyanobacteria, SSADH is an essential enzyme of its uncanonical tricarboxylic acid cycle. SSADH was also found to be able to oxidize glutarate semialdehyde, participating in lysine catabolism in bacteria.

			Action	DRS
	EPS-27-1.raw	Raw
	EPS-27-10.raw	Raw
	EPS-27-11.raw	Raw
	EPS-27-12.raw	Raw
	EPS-27-2.raw	Raw

Dataset Information

Identification of EcpK, a bacterial tyrosine pseudokinase important for exopolysaccharide biosynthesis in Myxococcus xanthus

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