RidA Proteins Prevent Metabolic Damage Inflicted by PLP-Dependent Dehydratases in All Domains of Life

Abstract

Pyridoxal 5′-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein of Salmonella enterica serovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were recently shown to be enamine deaminases. Here we demonstrate the damaging potential of enamines in the absence of RidA proteins. Notably, S. enterica strains lacking RidA have decreased activity of the PLP-dependent transaminase B enzyme IlvE, an enzyme involved in branched-chain amino acid biosynthesis. We reconstituted the threonine/serine dehydratase (IlvA)-dependent inhibition of IlvE in vitro, show that the in vitro system reflects the mechanism of RidA function in vivo, and show that IlvE inhibition is prevented by RidA proteins from all domains of life. We conclude that 2-aminoacrylate (2AA) inhibition represents a new type of metabolic damage, and this finding provides an important physiological context for the role of the ubiquitous RidA family of enamine deaminases in preventing damage by 2AA.

Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids

“…This assay is simpler and more suited to kinetic analysis than the end-point radioassay used previously for S. enterica proteins (Lambrecht et al, 2013). BCAT3 was chosen as the inactivation target based on its coexpression with RidA (see above), its similarity to S. enterica branched-chain aminotransferase IlvE, and its known role in branched-chain amino acid synthesis (Knill et al, 2008).…”

“…The members of the RidA family are small, sequence-diverse proteins that occur in all domains of life; prokaryote genomes often encode several of them (Lambrecht et al, 2013). Various phenotypes have been reported for ridA mutants in bacteria, yeast, and other organisms (Christopherson et al, 2012), and several crystal structures have been solved (Manjasetty et al, 2004;Burman et al, 2007;Pu et al, 2011).…”

“…The imine is then hydrolyzed to a 2-oxoacid, a reaction formerly thought to be purely spontaneous (Chargaff and Sprinson, 1943). The reactive enamine/imine tautomers (henceforth simply called imines) can attack other compounds before being hydrolyzed (Datta and Bhadra, 1978;Hillebrand et al, 1979), and in S. enterica, the imine formed from Ser attacks the pyridoxal 59-phosphate (PLP) cofactor of the Ile biosynthesis enzyme IlvE, a branched-chain aminotransferase (BCAT), forming an adduct that inactivates the enzyme (Figure 1) (Lambrecht et al, 2013). By rapidly hydrolyzing the imine, RidA preempts this damage to IlvE.…”

“…Damage preemption typically works by converting an aggressively reactive product into a more benign one, thereby forestalling damage; the Nudix family hydrolases that degrade noncanonical, genotoxic NTPs are classic examples (Bessman et al, 1996). Emerging evidence indicates that the RidA family of proteins (formerly called the YjgF/YER057c/UK114 family) is a new class of damage preemption enzymes (Lambrecht et al, 2013).…”

Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity

“…Facile decomposition of Thr would be a metabolic burden in vivo and the aminocrotonate produced by decomposition is highly toxic, and cells possess enzymes to accelerate hydrolysis of this metabolite. 15 Therefore, it seems unlikely that a significant proportion of TrpS specificity can be attributed to deamination of Thr. Instead, we hypothesize that allosteric effects are responsible for the increase in specificity when IGP is used instead of indole.…”