Glutamine Phosphoribosylpyrophosphate Amidotransferase-independent Phosphoribosyl Amine Synthesis from Ribose 5-Phosphate and Glutamine or Asparagine

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic L-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed

NLRP12 attenuates colon inflammation by maintaining colonic microbial diversity and promoting protective commensal bacterial growth

Inflammatory bowel diseases involve the dynamic interplay of host genetics, microbiome and inflammatory response. Here, we report that NLRP12, a negative regulator of innate immunity, is reduced in human ulcerative colitis by comparing monozygotic twins and other patient cohorts. In parallel, Nlrp12-deficiency in mice caused increased colonic basal inflammation, leading to a less-diverse microbiome, loss of protective gut commensal strains (Lachnospiraceae) and increased colitogenic strains (Erysipelotrichaceae). Dysbiosis and colitis susceptibility associated with Nlrp12-deficency were reversed equally by treatment with antibodies targeting inflammatory cytokines or by administration of beneficial commensal Lachnospiraceae isolates. Fecal transplants from specific pathogen free reared mice into germ-free Nlrp12-deficient mice showed that NLRP12 and the microbiome each contribute to immune signaling that culminates in colon inflammation. These findings reveal a feed-forward loop where NLRP12 promotes specific commensals that can reverse gut inflammation, while cytokine blockade during NLRP12-deficiency can reverse dysbiosis

Perturbations in histidine biosynthesis uncover robustness in the metabolic network of Salmonella enterica.

Phosphoribosylamine (PRA) is an intermediate in the biosynthetic pathway that is common to thiamine and purines. Glutamine phosphoribosyl pyrophosphate (PRPP) amidotransferase is the product of the purF gene in Salmonella enterica and catalyzes the synthesis of PRA from PRPP and glutamine. Strains lacking PurF require exogenous addition of purines for growth. However, under some growth conditions or with specific secondary mutations these strains grow in the absence of exogenous thiamine. Mutant alleles of hisA, which encodes 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide (ProFAR) isomerase, allowed PurF-independent PRA formation. The alleles of hisA that suppressed the requirement for exogenous thiamine resulted in proteins with reduced enzymatic activity. Data presented here showed that decreased activity of HisA altered metabolite pools and allowed PRA formation from ProFAR. Possible mechanisms of this conversion were proposed. The results herein emphasize the plasticity of the metabolic network and specifically highlight the potential for chemical syntheses to contribute to network robustness

HPLC separation of ProFAR breakdown products.

<p>The dashed line indicates the trace of stock 1 mM ProFAR used for this assay. The solid line (offset) indicates the trace of 1 mM ProFAR pH 7.5 after incubation at 37°C for 26 hours. Stars indicate unknown break down products. Abbreviations: AICAR, 5-amino-4-imidazolecarboxamide ribonucleotide; ProFAR, 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino]imidazole-4-carboxamide.</p

Growth rates of some <i>hisA</i> mutant strains are increased by exogenous histidine.

<p>Growth rates (in hours⁻¹) are shown and are arranged in descending order by their ability to generate their own histidine (middle data column). All strains were grown in minimal glucose medium at 37°C with adenine and the indicated additions. Thi: thiamine; His: histidine</p>a<p>NG = no growth; growth rate was <0.03 hours⁻¹.</p

Possible mechanisms for PRA formation from ProFAR.

<p>General mechanisms for PRA formation from ProFAR are depicted schematically. In pathway I, ProFAR is hydrolyzed to generate R5P by a mechanism that likely requires an enzyme. Ammonia is also released from the non-R5P product and is then available for non-enzymatic formation of PRA. It is possible the R5P and/or the ammonia do not leave the active site of the relevant enzyme. Pathway II depicts the formation of PRA as a direct product and implicates an undefined enzyme-catalyzed mechanism. Abbreviations: ProFAR, 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide; R5P, ribose-5′-phosphate; PRA, phosphoribosylamine.</p

Bacterial strains.

a<p>MudJ refers to the Mud1734 transposon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048207#pone.0048207-Castilho1" target="_blank">[9]</a>.</p>b<p>Allele numbers for <i>hisA</i> in the 1400 s were issued by the Salmonella Genetic Stock Center as <i>hsi</i> alleles for historical reasons. For simplicity, we have used the <i>his</i> designation herein.</p>c<p>Tn<i>10d</i>(Tc) refers to the transposition-defective mini-Tn<i>10</i>(Tn<i>10Δ16Δ17</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048207#pone.0048207-Way1" target="_blank">[10]</a>.</p

Diverse mutations in <i>hisA</i> allow PurF-independent PRA synthesis.

a<p>From the annotated LT2 genome, NCBI GeneID: 1253299. Numbering starts at the first nucleotide of the coding sequence for HisA. Δ: Deletion</p>b<p>Independent isolates.</p

Metabolic flux to ProFAR is required for PurF-independent PRA synthesis.

<p>Growth rates (in hours⁻¹) are shown. Strains were grown in minimal glucose medium with adenine and the indicated additions. His: histidine; Thi: thiamine. Histidine alleles <i>hisA3000, hisI99, hisF109, his-2652</i> (<i>del:CBHAFI</i>) cause a complete loss of function of the relevant gene product(s). Allele <i>hisG1102</i> encodes an enzyme that is insensitive to feedback inhibition by histidine.</p>a<p>NG = no growth; growth rate was <0.03 hours⁻¹.</p