PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa

Abstract

The Rid protein superfamily (YjgF/YER057c/UK114) is found in all domains of life. The archetypal protein, RidA from , is a deaminase that quenches the reactive metabolite 2-aminoacrylate (2AA). 2AA deaminase activity is conserved in RidA proteins from humans, plants, yeast, archaea, and bacteria. Mutants of, , and that lack a functional RidA exhibit growth defects, suggesting that 2AA metabolic stress is similarly conserved. The PubSEED database shows (PAO1) encodes eight members of the Rid superfamily. Mutants of PAO1 lacking each of five Rid proteins were screened, and the mutant phenotypes that arose in the absence of PA5339 were dissected. A PA5339::Tn mutant has growth, motility, and biofilm defects that can all be linked to the accumulation of 2AA. Further, the PA5339 protein was demonstrably a 2AA deaminase and restored metabolic balance to a mutant The data presented here show that the RidA paradigm in had similarities to those described in other organisms but was distinct in that deleting only one of multiple homologs generated deficiencies. Based on the collective data presented here in, PA5339 was renamed RidA. RidA is a widely conserved protein that prevents endogenous metabolic stress caused by 2-aminoacrylate (2AA) damage to pyridoxal 5'-phosphate (PLP)-dependent enzymes in prokaryotes and eukaryotes. The framework for understanding the accumulation of 2AA and its consequences have largely been defined in We show here that in (PAO1), 2AA accumulation leads to reduced growth, compromised motility, and defective biofilm formation. This study expands our knowledge how the metabolic architecture of an organism contributes to the consequences of 2AA inactivation of PLP-dependent enzymes and identifies a key RidA protein in .

Cj1388 Is a RidA Homolog and Is Required for Flagella Biosynthesis and/or Function in Campylobacter jejuni

“…The RidA, r eactive i ntermediate d eaminase A , of S. enterica was found to be an enamine deaminase, and multiple homologs from the three domains of life have similar activity (Lambrecht et al, 2010; Lambrecht et al, 2012; Niehaus et al, 2015; ElRamlawy et al, 2016; Ernst and Downs, 2018). In some organisms, a cellular role for RidA involves quenching the reactive metabolite 2-aminoacrylate (2AA) to prevent damage to specific pyridoxal 5′-phosphate (PLP)-dependent enzymes (Schmitz and Downs, 2004; Christopherson et al, 2008; Flynn and Downs, 2013; Flynn et al, 2013; Lambrecht et al, 2013; Ernst et al, 2014, 2016; Niehaus et al, 2015; Irons et al, 2018). RidA homologs have a similar role in at least Escherichia coli , Pseudomonas aeruginosa , and Saccharomyces cerevisiae , although the phenotypic consequences of a ridA mutation depends on the specific metabolic network architecture of the organism (Borchert and Downs, 2017b; Ernst and Downs, 2018; Irons et al, 2018).…”

“…In some organisms, a cellular role for RidA involves quenching the reactive metabolite 2-aminoacrylate (2AA) to prevent damage to specific pyridoxal 5′-phosphate (PLP)-dependent enzymes (Schmitz and Downs, 2004; Christopherson et al, 2008; Flynn and Downs, 2013; Flynn et al, 2013; Lambrecht et al, 2013; Ernst et al, 2014, 2016; Niehaus et al, 2015; Irons et al, 2018). RidA homologs have a similar role in at least Escherichia coli , Pseudomonas aeruginosa , and Saccharomyces cerevisiae , although the phenotypic consequences of a ridA mutation depends on the specific metabolic network architecture of the organism (Borchert and Downs, 2017b; Ernst and Downs, 2018; Irons et al, 2018). Enzyme damage resulting from accumulated 2AA can impact growth, motility, biofilm formation, iron homeostasis, and potentially virulence.…”

“…In the absence of RidA, free 2AA accumulates and can covalently inactivate certain PLP-dependent enzymes such as serine hydroxymethyltransferase (SHMT) (GlyA; EC 2.1.2.1), alanine racemases (Alr/DadX; EC 5.1.1.1), and transaminase B (IlvE; EC 2.6.1.42), leading to defects in one-carbon unit metabolism, cell-wall synthesis, and isoleucine biosynthesis, respectively (Schmitz and Downs, 2004; Flynn et al, 2013; Ernst and Downs, 2016). The activity of the enzymes targeted by 2AA can be decreased by 30–50% in strains lacking RidA (Lambrecht et al, 2010; Ernst and Downs, 2016, 2018; Borchert and Downs, 2017b; Irons et al, 2018). To date, the diverse phenotypes of organisms lacking RidA suggest that there are additional and unknown targets of 2AA, possibly extending beyond PLP-dependent enzymes.…”

“…In at least S. enterica , E. coli , P. aeruginosa , S. cerevisiae , and Arabidopsis thaliana , a biosynthetic threonine/serine dehydratase (IlvA), acting on serine is the main source of 2AA in ridA mutants (Lambrecht et al, 2012; Niehaus et al, 2014; Borchert and Downs, 2017a; Ernst and Downs, 2018; Irons et al, 2018). In each of these organisms, IlvA has a regulatory domain and is allosterically inhibited by isoleucine (Gallagher et al, 1998; Schmitz and Downs, 2004).…”

“…The specific targets of 2AA that result in detectable defects vary in different organisms. In S. enterica , 2AA accumulation causes a growth defect reversed by exogenous glycine; in E. coli 2AA accumulation-induced growth inhibition was reversed by exogenous aspartate, or purines; in P. aeruginosa , 2AA accumulation is detrimental to growth and partially reversed by exogenous proline and polyamines; and in S. cerevisiae mitochondrial accumulation of 2AA leads to loss of mitochondrial DNA and reduced heme biosynthesis (Flynn et al, 2013; Ernst and Downs, 2016, 2018; Borchert and Downs, 2017b; Irons et al, 2018) (Whitaker and Downs, unpublished). The diverse effects of 2AA emphasize the complexity of the metabolic network and our limited understanding of the integration between biochemical pathways.…”