Decreased coenzyme A levels in ridA mutant strains of Salmonella enterica result from inactivated serine hydroxymethyltransferase

Abstract

Summary The RidA/Yer057/UK114 family of proteins is well represented across the domains of life and recent work has defined both an in vitro activity and an in vivo role for RidA. RidA proteins have enamine deaminase activity, and in their absence the reactive 2-aminoacrylate (2-AA) accumulates and inactivates at least some pyridoxal 5′-phosphate (PLP)-containing enzymes in Salmonella enterica. The conservation of RidA suggested that 2-AA was a ubiquitous cellular stressor that was generated in central metabolism. Phenotypically, strains of S. enterica that lack RidA accumulated significantly more pyruvate in the growth medium than wild-type strains. Here we dissected this ridA mutant phenotype and showed it was an indirect consequence of damage to serine hydroxymethyltransferase (GlyA; E.C. 2.1.2.1). The results here identified a fourth PLP enzyme as a target of enamine stress in Salmonella.

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

“…The CoA precursor pantothenate stimulated CoA production in a ridA strain but failed to restore wild-type growth. In contrast, glycine restored both growth and CoA production to wild-type levels in a ridA strain grown in minimal glucose medium (4). Building on this previous work, results presented herein demonstrate that glycine alleviates all ridA-dependent growth defects.…”

“…The effect exogenous glycine had on growth of a ridA strain in minimal glucose medium was reported previously (4). Glycine suppressed the minor growth defect of a ridA strain and prevented pyruvate accumulation, suggesting that glycine limitation may contribute to alternative ridA phenotypes.…”

“…In S. enterica, RidA is known to preempt metabolic damage caused by the unbound reactive enamine 2-aminoacrylate (2AA) (2). In the absence of RidA, 2AA persisted in solution long enough to inactivate multiple target enzymes in vivo and in vitro (2)(3)(4). RidA proteins isolated from organisms spanning the tree of life displayed similar enamine and imine deaminase activities (5,6), suggesting that RidA enzymes have a conserved role in preventing 2AA stress.…”

“…In the absence of exogenous 2AA precursors, basal IlvA activity produced sufficient 2AA from endogenous serine to cause a minor growth defect in ridA strains grown in minimal glucose medium (9). The minor growth defect coincided with measurable 2AA inactivation of several PLP-dependent target enzymes, including 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) (2)(3)(4). Isoleucine prevented IlvA-dependent 2AA production from serine (endogenous or exogenous) and restored growth of ridA strains to wild-type levels through feedback inhibition of IlvA (9).…”

“…Of the 2AA targets described to date, GlyA was inactivated to the greatest extent in ridA strains relative to the wild type (20% of wild-type activity in minimal glucose medium) (4). GlyA catalyzes the reversible transfer of a hydroxymethyl group from serine to tetrahydrofolate (THF), forming glycine and the one-carbon unit 5,10-methylene-tetrahydrofolate (5,10-mTHF) (Fig.…”