Complex Metabolic Phenotypes Caused by a Mutation in yjgF,Encoding a Member of the Highly Conserved YER057c/YjgF Family of Proteins

The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in Salmonella typhimurium. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.The increasing number of completed genome sequences has resulted in the identification of new families of hypothetical proteins whose function has yet to be established. The lack of existing mutants defective in these conserved proteins suggests novel, complex, or subtle phenotypes. Through our work on thiamine synthesis in Salmonella typhimurium, we have isolated mutants defective in the recently identified YER057c/YjgF protein family. Our data suggest that defects in this protein result in complex phenotypes involving thiamine and isoleucine biosynthesis.Thiamine pyrophosphate (TPP) serves as an essential cofactor for a number of metabolic reactions involving the removal or transfer of C₂ units. Despite the important role of TPP in cellular metabolism, its synthesis and regulation are not well understood in any organism. TPP is formed from two precursors, 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (THZ-P) and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP). These compounds are joined and subsequently phosphorylated as shown in Fig. ​Fig.1A.1A. Although many of the enzymatic steps in both the THZ-P and HMP-PP pathways have not been clearly defined, the major precursor molecules for both of these compounds have been determined by labeling studies (17, 20, 28, 29). In particular, the purine pathway intermediate, aminoimidazole ribotide (AIR), has been shown to provide all of the atoms in HMP (28, 50, 51).Open in a separate windowFIG. 1Pathway schematics. (A) Biosynthetic pathway for TPP. The involvement of the purine pathway in HMP-PP synthesis is shown with structural intermediates prior to the AIR branch point. Arrows denoted with dotted lines represent proposed steps. Reactions involved in the conversion of AIR to HMP-PP and in the synthesis of THZ-P have not been clearly defined. Genes whose products are required for selected reactions are indicated next to the relevant arrows. Abbreviations: R-P, ribose-5-phosphate, PRPP, phosphoribosylpyrophosphate. (B) Biosynthetic pathways for the branched-chain amino acids isoleucine and valine. Enzymes that catalyze specific steps are as follows: 1, aspartate transaminase; 2, 3, and 4, aspartate kinases I, II, and III, respectively; 5, aspartate semialdehyde dehydrogenase; 6 and 7, homoserine dehydrogenases I and II, respectively; 8, homoserine kinase; 9, threonine synthase; 10, threonine deaminase; 11 and 12, acetohydroxy acid synthases I and II, respectively; 13, acetohydroxy acid isomeroreductase; 14, dihydroxy acid dehydratase; 15, transaminase B; 16, transaminase C. OAA, oxaloacetic acid.Although the involvement of the purine pathway in the synthesis of HMP is clear, there is substantial genetic and biochemical evidence indicating that the first enzyme of the purine pathway, phosphoribosylpyrophosphate amidotransferase (PurF) (EC 2.4.2.14), is not required for HMP synthesis in S. typhimurium under all conditions. Mutants defective in purF are able to grow in the absence of thiamine when glucose is used as a carbon source if pantothenate is also supplied in the medium (23). Similarly, purF mutants do not require thiamine when grown on a number of nonglucose carbon sources, such as gluconate or ribose (54). The pathway responsible for synthesis of HMP independent of the PurF enzyme has been defined as the alternative pyrimidine biosynthetic (APB) pathway (21, 54); recent biochemical data suggest that phosphoribosylamine (PRA), or a derivative, is an intermediate in this pathway (24).Significant progress in our understanding of the APB pathway has been made by the isolation and characterization of mutants unable to synthesize thiamine in a purF background. One class of mutants, designated apbA, was defective in a pantothenate biosynthetic enzyme (ketopantoate reductase [PanE]) (32, 33), consistent with previous results implicating a role for pantothenate in PurF-independent thiamine synthesis (23). A second class of these mutants was defective in the oxidative pentose phosphate pathway, affecting either glucose-6-phosphate dehydrogenase (Zwf) or gluconate-6-phosphate dehydrogenase (Gnd) (25, 54). Addition of ribose-5-phosphate (ribose-5-P) restored function of the APB pathway in these mutants, suggesting that the role of these enzymes in HMP synthesis was to supply ribose-5-P. These results led to the model shown in Fig. ​Fig.1A1A which implicates ribose-5-P and an amine donor as precursors to PRA. Repeated attempts have failed to identify either the predicted PRA-forming activity or mutants defective in this step (27). There are several possible explanations for this. It is possible that the correct substrates have not been identified and/or that the PRA-forming activity is required for another cellular function.In this report, we describe the isolation and characterization of mutations that allow function of the APB pathway in the absence of the pentose phosphate pathway. These mutations were found to disrupt a previously uncharacterized open reading frame (ORF) encoding a hypothetical 13.5-kDa protein. We have designated this gene yjgF based on homology to the respective ORF in Escherichia coli. The YjgF protein belongs to the YER057c/YjgF protein family, a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms. Characterization of these mutants revealed that they also were sensitive to the presence of serine in the medium, exhibiting a requirement for isoleucine under this condition. The phenotypes caused by yjgF mutations suggest that the YjgF protein may be involved in regulation or function of the isoleucine biosynthetic pathway. Further, results suggest a connection between isoleucine biosynthesis and function of the APB pathway in thiamine synthesis.