Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family.

BackgroundIt is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5'-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.ResultsPhylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5'-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.ConclusionsLike the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5'-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious

Genomic and Experimental Evidence for Multiple Metabolic Functions in the RidA/YjgF/YER057c/UK114 (Rid) Protein Family

BACKGROUND: It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5\u27-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions. RESULTS: Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5\u27-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate. CONCLUSIONS: Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5\u27-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious

Innovations in asthma therapy: is there a role for inhaled statins?

INTRODUCTION:Asthma manifests as chronic airflow obstruction with persistent inflammation and airway hyperresponsiveness. The immunomodulatory and anti-inflammatory properties of the HMG-CoA reductase (HMGCR) inhibitors (a.k.a. statins), suggest a therapeutic role in chronic inflammatory lung diseases. However, despite positive laboratory investigations and promising epidemiological data, clinical trials using statins for the treatment of asthma have yielded conflicting results. Inadequate statin levels in the airway compartment could explain these findings. Areas covered: HMGCR is in the mevalonate (MA) pathway and MA signaling is fundamental to lung biology and asthma. This article will discuss clinical trials of oral statins in asthma, review lab investigations relevant to the systemic versus inhaled administration of statins, address the advantages and disadvantages of inhaled statins, and answer the question: is there a role for inhaled statins in the treatment of asthma? Expert commentary: If ongoing investigations show that oral administration of statins has no clear clinical benefits, then repurposing statins for delivery via inhalation is a logical next step. Inhalation of statins bypasses first-pass metabolism by the liver, and therefore, allows for delivery of significantly lower doses to the airways at greater potency. Statins could become the next major class of novel inhalers for the treatment of asthma

Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight

5-Oxoproline (OP) is well-known as an enzymatic intermediate in the eukaryotic γ-glutamyl cycle, but it is also an unavoidable damage product formed spontaneously from glutamine and other sources. Eukaryotes metabolize OP via an ATP-dependent 5-oxoprolinase; most prokaryotes lack homologs of this enzyme (and the γ-glutamyl cycle) but are predicted to have some way to dispose of OP if its spontaneous formation in vivo is significant. Comparative analysis of prokaryotic genomes showed that the gene encoding pyroglutamyl peptidase, which removes N-terminal OP residues, clusters in diverse genomes with genes specifying homologs of a fungal lactamase (renamed prokaryotic 5-oxoprolinase A, pxpA) and homologs of allophanate hydrolase subunits (renamed pxpB and pxpC). Inactivation of Bacillus subtilis pxpA, pxpB, or pxpC genes slowed growth, caused OP accumulation in cells and medium, and prevented use of OP as a nitrogen source. Assays of cell lysates showed that ATP-dependent 5-oxoprolinase activity disappeared when pxpA, pxpB, or pxpC was inactivated. 5-Oxoprolinase activity could be reconstituted in vitro by mixing recombinant B. subtilis PxpA, PxpB, and PxpC proteins. In addition, overexpressing Escherichia coli pxpABC genes in E. coli increased 5-oxoprolinase activity in lysates ≥1700-fold. This work shows that OP is a major universal metabolite damage product and that OP disposal systems are common in all domains of life. Furthermore, it illustrates how easily metabolite damage and damage-control systems can be overlooked, even for central metabolites in model organisms

Tracking Polymicrobial Metabolism in Cystic Fibrosis Airways: Pseudomonas aeruginosa Metabolism and Physiology Are Influenced by Rothia mucilaginosa-Derived Metabolites.

Due to a lack of effective immune clearance, the airways of cystic fibrosis patients are colonized by polymicrobial communities. One of the most widespread and destructive opportunistic pathogens is Pseudomonas aeruginosa; however, P. aeruginosa does not colonize the airways alone. Microbes that are common in the oral cavity, such as Rothia mucilaginosa, are also present in cystic fibrosis patient sputum and have metabolic capacities different from those of P. aeruginosa Here we examine the metabolic interactions of P. aeruginosa and R. mucilaginosa using stable-isotope-assisted metabolomics. Glucose-derived 13C was incorporated into glycolysis metabolites, namely, lactate and acetate, and some amino acids in R. mucilaginosa grown aerobically and anaerobically. The amino acid glutamate was unlabeled in the R. mucilaginosa supernatant but incorporated the 13C label after P. aeruginosa was cross-fed the R. mucilaginosa supernatant in minimal medium and artificial-sputum medium. We provide evidence that P. aeruginosa utilizes R. mucilaginosa-produced metabolites as precursors for generation of primary metabolites, including glutamate.IMPORTANCEPseudomonas aeruginosa is a dominant and persistent cystic fibrosis pathogen. Although P. aeruginosa is accompanied by other microbes in the airways of cystic fibrosis patients, few cystic fibrosis studies show how P. aeruginosa is affected by the metabolism of other bacteria. Here, we demonstrate that P. aeruginosa generates primary metabolites using substrates produced by another microbe that is prevalent in the airways of cystic fibrosis patients, Rothia mucilaginosa These results indicate that P. aeruginosa may get a metabolic boost from its microbial neighbor, which might contribute to its pathogenesis in the airways of cystic fibrosis patients

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

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase

Tracking Polymicrobial Metabolism in Cystic Fibrosis Airways: Pseudomonas aeruginosa Metabolism and Physiology Are Influenced by Rothia mucilaginosa-Derived Metabolites.

Due to a lack of effective immune clearance, the airways of cystic fibrosis patients are colonized by polymicrobial communities. One of the most widespread and destructive opportunistic pathogens is Pseudomonas aeruginosa; however, P. aeruginosa does not colonize the airways alone. Microbes that are common in the oral cavity, such as Rothia mucilaginosa, are also present in cystic fibrosis patient sputum and have metabolic capacities different from those of P. aeruginosa Here we examine the metabolic interactions of P. aeruginosa and R. mucilaginosa using stable-isotope-assisted metabolomics. Glucose-derived 13C was incorporated into glycolysis metabolites, namely, lactate and acetate, and some amino acids in R. mucilaginosa grown aerobically and anaerobically. The amino acid glutamate was unlabeled in the R. mucilaginosa supernatant but incorporated the 13C label after P. aeruginosa was cross-fed the R. mucilaginosa supernatant in minimal medium and artificial-sputum medium. We provide evidence that P. aeruginosa utilizes R. mucilaginosa-produced metabolites as precursors for generation of primary metabolites, including glutamate.IMPORTANCEPseudomonas aeruginosa is a dominant and persistent cystic fibrosis pathogen. Although P. aeruginosa is accompanied by other microbes in the airways of cystic fibrosis patients, few cystic fibrosis studies show how P. aeruginosa is affected by the metabolism of other bacteria. Here, we demonstrate that P. aeruginosa generates primary metabolites using substrates produced by another microbe that is prevalent in the airways of cystic fibrosis patients, Rothia mucilaginosa These results indicate that P. aeruginosa may get a metabolic boost from its microbial neighbor, which might contribute to its pathogenesis in the airways of cystic fibrosis patients

Tracking Polymicrobial Metabolism in Cystic Fibrosis Airways: Pseudomonas aeruginosa Metabolism and Physiology Are Influenced by Rothia mucilaginosa-Derived Metabolites

ABSTRACT Due to a lack of effective immune clearance, the airways of cystic fibrosis patients are colonized by polymicrobial communities. One of the most widespread and destructive opportunistic pathogens is Pseudomonas aeruginosa; however, P. aeruginosa does not colonize the airways alone. Microbes that are common in the oral cavity, such as Rothia mucilaginosa, are also present in cystic fibrosis patient sputum and have metabolic capacities different from those of P. aeruginosa. Here we examine the metabolic interactions of P. aeruginosa and R. mucilaginosa using stable-isotope-assisted metabolomics. Glucose-derived 13C was incorporated into glycolysis metabolites, namely, lactate and acetate, and some amino acids in R. mucilaginosa grown aerobically and anaerobically. The amino acid glutamate was unlabeled in the R. mucilaginosa supernatant but incorporated the 13C label after P. aeruginosa was cross-fed the R. mucilaginosa supernatant in minimal medium and artificial-sputum medium. We provide evidence that P. aeruginosa utilizes R. mucilaginosa-produced metabolites as precursors for generation of primary metabolites, including glutamate. IMPORTANCE Pseudomonas aeruginosa is a dominant and persistent cystic fibrosis pathogen. Although P. aeruginosa is accompanied by other microbes in the airways of cystic fibrosis patients, few cystic fibrosis studies show how P. aeruginosa is affected by the metabolism of other bacteria. Here, we demonstrate that P. aeruginosa generates primary metabolites using substrates produced by another microbe that is prevalent in the airways of cystic fibrosis patients, Rothia mucilaginosa. These results indicate that P. aeruginosa may get a metabolic boost from its microbial neighbor, which might contribute to its pathogenesis in the airways of cystic fibrosis patients

Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of 'free' (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6