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Diana M Downs

Publications and source records attributed to Diana M Downs.

18 recordsLinked to original sources
PubMed2006

Inhibition of fructose-1,6-bisphosphatase by aminoimidazole carboxamide ribotide prevents growth of Salmonella enterica purH mutants on glycerol.

The enzyme fructose-1,6-bisphosphatase (FBP) is key regulatory point in gluconeogenesis. Mutants of Salmonella enterica lacking purH accumulate 5-amino-4-imidazole carboxamide ribotide (AICAR) and are unable to utilize glycerol as sole carbon and energy sources. The work described here demonstrates this lack of growth is due to inhibition of FBP by AICAR. Mutant alleles of fbp that restore growth on glycerol encode proteins resistant to inhibition by AICAR and the allosteric regulator AMP. This is the first report of biochemical characterization of substitutions causing AMP resistance in a bacterial FBP. Inhibition of FBP activity by AICAR occurs at physiologically relevant concentrations and may represent a form of regulation of gluconeogenic flux in Salmonella enterica.

Adenosine Monophosphate↗
PubMed2006

Solution structure of YggX: a prokaryotic protein involved in Fe(II) trafficking.

We report the three-dimensional structure of YggX from Salmonella enterica, determined by solution nuclear magnetic resonance (NMR) spectroscopy from protein labeled with carbon-13 and nitrogen-15 produced by Escherichia coli cells. The protein has a beta1beta2alpha1alpha2alpha3 fold that is unique to YggX and one of its homologs, a protein from Pseudomonas aeruginosa with 45% sequence identity whose X-ray structure [Protein Data Bank (PDB) 1T07] was determined by a structural genomics center. The NMR structure, which revealed that the C-terminal region of YggX is dynamically disordered, explains why electron density from the corresponding region was missing in the X-ray structure of the Pseudomonas protein. Because it has been hypothesized that YggX has a role in iron trafficking, we investigated the influence of Fe(II) on the (1)H-(15)N NMR fingerprint region of nitrogen-15-labeled YggX. Several signals shifted or broadened upon the addition of excess Fe(II) under anoxic conditions, with His81 showing the largest effect. These results indicate that Fe(II) binds weakly to this protein at a region of the sequence conserved only in the subset of the YggX proteins from organisms similar to Salmonella. The finding that iron binds only weakly to YggX, and not to a highly conserved region of the structure, suggests that the role of this protein in iron homeostasis is more complex than previously thought.

Amino Acid Sequence↗
PubMed2006

A connection between iron-sulfur cluster metabolism and the biosynthesis of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate in Salmonella enterica.

Several cellular pathways have been identified which affect the efficiency of thiamine biosynthesis in Salmonella enterica. Mutants defective in iron-sulfur (Fe-S) cluster metabolism are less efficient at synthesis of the pyrimidine moiety of thiamine. These mutants are compromised for the conversion of aminoimidazole ribotide (AIR) to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), not the synthesis of AIR. The gene product ThiC contains potential ligands for an Fe-S cluster that are required for function in vivo. The conversion of AIR to HMP-P is sensitive to oxidative stress, and variants of ThiC have been identified that have increased sensitivity to oxidative growth conditions. The data are consistent with ThiC or an as-yet-unidentified protein involved in HMP-P synthesis containing an Fe-S cluster required for its physiological function.

Amino Acid Sequence↗
PubMed2006

PurF-independent phosphoribosyl amine formation in yjgF mutants of Salmonella enterica utilizes the tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase.

In Salmonella enterica, the biosynthetic pathways for the generation of purines and the essential cofactor thiamine pyrophosphate branch after sharing five enzymatic steps. Phosphoribosyl amine (PRA) is the first intermediate in the common portion of the pathway and is generated from phosphoribosylpyrophosphate and glutamine by the PurF enzyme (phosphoribosylpyrophosphate amidotransferase). A null mutation in yjgF allows PurF-independent PRA formation by an unknown mechanism. The tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase, composed of the TrpD and TrpE proteins, was shown to be essential for PRA formation in strains lacking both yjgF and purF. The activity generating PRA in a yjgF mutant background has features that distinguish it from the TrpDE-mediated PRA formation shown previously for this enzyme in strains with an active copy of yjgF. The data presented here are consistent with a model in which the absence of YjgF uncovers a new catalytic activity of TrpDE.

Anthranilate Phosphoribosyltransferase↗
PubMed2006

An allele of gyrA prevents Salmonella enterica serovar Typhimurium from using succinate as a carbon source.

A mutant gyrA allele resulting in an A271E substitution in the DNA gyrase protein generated a strain unable to grow on the C(4)-dicarboxylates succinate, malate, and fumarate. Bacteria harboring gyrA751 displayed decreased negative supercoiling in cells. Expression of the dctA gene, which encodes the C(4)-dicarboxylate transporter, was reduced in a gyrA751 mutant, providing the first evidence that dctA expression is supercoiling sensitive and uncovering a simple metabolic screen for lesions in gyrase that reduce negative supercoiling.

Alleles↗
PubMed2006

Understanding microbial metabolism.

Metabolism encompasses the biochemical basis of life and as such spans all biological disciplines. Many decades of basic research, primarily in microbes, have resulted in extensive characterization of metabolic components and regulatory paradigms. With this basic knowledge in hand and the technologies currently available, it has become feasible to move toward an understanding of microbial metabolism as a system rather than as a collection of component parts. Insight into the system will be generated by continued efforts to rigorously define metabolic components combined with renewed efforts to discover components and connections using in vivo-driven approaches. On the tail of a detailed understanding of components and connections that comprise metabolism will come the ability to generate a comprehensive mathematical model that describes the system. While microbes provide the logical organism for this work, the value of such a model would span biological disciplines. Described herein are approaches that can provide insight into metabolism and caveats of their use. The goal of this review is to emphasize that in silico, in vitro, and in vivo approaches must be used in combination to achieve a full understanding of microbial metabolism.

Bacteria↗
PubMed2004

Mutational analysis of ThiH, a member of the radical S-adenosylmethionine (AdoMet) protein superfamily.

Thiamine pyrophosphate (TPP) is an essential cofactor for all forms of life. In Salmonella enterica, the thiH gene product is required for the synthesis of the 4-methyl-5-beta hydroxyethyl-thiazole monophosphate moiety of TPP. ThiH is a member of the radical S-adenosylmethionine (AdoMet) superfamily of proteins that is characterized by the presence of oxygen labile [Fe-S] clusters. Lack of an in vitro activity assay for ThiH has hampered the analysis of this interesting enzyme. We circumvented this problem by using an in vivo activity assay for ThiH. Random and directed mutagenesis of the thiH gene was performed. Analysis of auxotrophic thiH mutants defined two classes, those that required thiazole to make TPP (null mutants) and those with thiamine auxotrophy that was corrected by either L-tyrosine or thiazole (ThiH* mutants). Increased levels of AdoMet also corrected the thiamine requirement of members of the latter class. Residues required for in vivo function were identified and are discussed in the context of structures available for AdoMet enzymes.

Alleles↗
PubMed2004

Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo.

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

Alanine↗
PubMed2004

Crystal structure of an aminoimidazole riboside kinase from Salmonella enterica: implications for the evolution of the ribokinase superfamily.

The crystal structures of a Salmonella enterica aminoimidazole riboside (AIRs) kinase, its complex with the substrate AIRs, and its complex with AIRs and an ATP analog were determined at 2.6 angstroms, 2.9 angstroms, and 2.7 angstroms, respectively. The product of the Salmonella-specific gene stm4066, AIRs kinase, is a homodimer with one active site per monomer. The core structure, consisting of an eight-stranded beta sheet flanked by eight alpha helices, indicates that AIRs kinase is a member of the ribokinase superfamily. Unlike ribokinase and adenosine kinase in this superfamily, AIRs kinase does not show significant conformational changes upon substrate binding. The active site is covered by a lid formed by residues 16-28 and 86-100. A comparison of the structure of AIRs kinase with other ribokinase superfamily members suggests that the active site lid and conformational changes that occur upon substrate binding may be advanced features in the evolution of the ribokinase superfamily.

Amines↗
PubMed2004

A mutant allele of rpoD results in increased conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine in Salmonella enterica.

An allele of rpoD (rpoD1181) that results in increased synthesis of the pyrimidine moiety of thiamine in Salmonella enterica was identified. The S508Y substitution caused by rpoD1181 is analogous to the S506F derivative of the Escherichia coli protein. The properties of this E. coli mutant protein have been well characterized in vitro. Identification of a metabolic phenotype caused by the rpoD1181 allele of S. enterica allows past in vitro results to be incorporated in continuing efforts to understand cellular processes that are integrated with the thiamine biosynthetic pathway.

Alleles↗
PubMed2004

Reduced transaminase B (IlvE) activity caused by the lack of yjgF is dependent on the status of threonine deaminase (IlvA) in Salmonella enterica serovar Typhimurium.

The YjgF/YER057c/UK114 family is a highly conserved class of proteins that is represented in the three domains of life. Thus far, a biochemical function demonstrated for these proteins in vivo or in vitro has yet to be defined. In several organisms, strains lacking a YjgF homolog have a defect in branched-chain amino acid biosynthesis. This study probes the connection between yjgF and isoleucine biosynthesis in Salmonella enterica. In strains lacking yjgF the specific activity of transaminase B, catalyzing the last step in the synthesis of isoleucine, was reduced. In the absence of yjgF, transaminase B activity could be restored by inhibiting threonine deaminase, the first enzymatic step in isoleucine biosynthesis. Strains lacking yjgF showed an increased sensitivity to sulfometruron methyl, a potent inhibitor of acetolactate synthase. Based on work described here and structural reports in the literature, we suggest a working model in which YjgF has a role in protecting the cell from toxic effects of imbalanced ketoacid pools.

Bacterial Proteins↗
PubMed2003

The YggX protein of Salmonella enterica is involved in Fe(II) trafficking and minimizes the DNA damage caused by hydroxyl radicals: residue CYS-7 is essential for YggX function.

Previous work from our laboratory identified YggX as a protein whose accumulation increased the resistance of Salmonella enterica to superoxide stress, reversed defects attributed to oxidized [Fe-S] clusters, and decreased the spontaneous mutation frequency of the cells. Here we present work aimed at determining why the accumulation of YggX correlates with reduced mutation frequency. Genetic and biochemical data showed that accumulation of YggX reduced the damage to DNA by hydroxyl radicals. The ability of purified YggX to protect DNA from Fenton chemistry mediated damage in vitro and to decrease the concentration of Fe(II) ions in solution available for chelation provided a framework for the interpretation of data obtained from in vivo experiments. The interpretation of in vitro assay results, within the context of the in vivo phenotypes, was validated by a mutant variant of YggX (C7S) that was unable to function in vivo or in vitro. We propose a model, based on data presented here and reported earlier, that suggests YggX is a player in Fe(II) trafficking in bacteria.

Bacterial Proteins↗
PubMed2003

The stm4066 gene product of Salmonella enterica serovar Typhimurium has aminoimidazole riboside (AIRs) kinase activity and allows AIRs to satisfy the thiamine requirement of pur mutant strains.

In bacteria the biosynthetic pathways for purine mononucleotides and the hydroxymethyl pyrimidine moiety of thiamine share five reactions that result in the formation of aminoimidazole ribotide, the last metabolite common to both pathways. Here we describe the characterization of a Salmonella enterica mutant strain that has gained the ability to efficiently use exogenous aminoimidazole riboside (AIRs) as a source of thiamine. The lesion responsible for this phenotype is a null mutation in a transcriptional regulator of the GntR family (encoded by stm4068). Lack of this protein derepressed transcription of an associated operon (stm4065-4067) that encoded a predicted kinase. The stm4066 gene product was purified and shown to have AIRs kinase activity in vitro. This activity was consistent with the model presented to explain the phenotype caused by the original mutation. This mutation provides a genetic means to isolate the synthesis of the hydroxymethyl pyrimidine moiety of thiamine from the pathway for purine mononucleotide biosynthesis and thus facilitate in vivo analyses.

Amino Acid Sequence↗
PubMed2003

Lack of the ApbC or ApbE protein results in a defect in Fe-S cluster metabolism in Salmonella enterica serovar Typhimurium.

The isc genes function in the assembly of Fe-S clusters and are conserved in many prokaryotic and eukaryotic organisms. In most bacteria studied, the isc operon can be deleted without loss of cell viability, indicating that additional systems for Fe-S cluster assembly must exist. Several laboratories have described nutritional and biochemical defects resulting from mutations in the isc operon. Here we demonstrate that null mutations in two genes of unknown function, apbC and apbE, result in similar cellular deficiencies. Exogenous ferric chloride suppressed these deficiencies in the apbC and apbE mutants, distinguishing them from previously described isc mutants. The deficiencies caused by the apbC and isc mutations were additive, which is consistent with Isc and ApbC's having redundant functions or with Isc and ApbC's functioning in different areas of Fe-S cluster metabolism (e.g., Fe-S cluster assembly and Fe-S cluster repair). Both the ApbC and ApbE proteins are similar in sequence to proteins that function in metal cofactor assembly. Like the enzymes with sequence similarity to ApbC, purified ApbC protein was able to hydrolyze ATP. The data herein are consistent with the hypothesis that the ApbC and ApbE proteins function in Fe-S cluster metabolism in vivo.

Amino Acid Sequence↗
PubMed2003

Anthranilate synthase can generate sufficient phosphoribosyl amine for thiamine synthesis in Salmonella enterica.

In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PurF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.

Amidophosphoribosyltransferase↗
PubMed2003

Genomics and bacterial metabolism.

The field of bacterial metabolism and physiology is arguably the oldest in microbiology. Much of our understanding of biological processes and molecular paradigms has its roots In early studies of prokaryotic physiology. After a period of declining interest in metabolic studies (prompted by the insurgence of molecular techniques), genomic technologies are revitalizing the study of bacterial metabolism and physiology. These new technologies bring a means to approach metabolic questions with a global perspective. When used in combination with classical and molecular techniques, emerging global technologies will make it feasible to understand the complex integration of metabolic processes that result in an efficient physiology. At the same time, without increased computational capabilities, the massive amounts of data generated by these technologies threaten to overwhelm, rather than facilitate, this work. For genomic technologies to reach their potential for increasing our understanding of bacterial metabolism, microbiologists must become more collaborative and multidisciplinary than at any time in our history.

Bacteria↗
PubMed2002

Metabolic flux in both the purine mononucleotide and histidine biosynthetic pathways can influence synthesis of the hydroxymethyl pyrimidine moiety of thiamine in Salmonella enterica.

Together, the biosyntheses of histidine, purines, and thiamine pyrophosphate (TPP) contain examples of convergent, divergent, and regulatory pathway integration. Mutations in two purine biosynthetic genes (purI and purH) affect TPP biosynthesis due to flux through the purine and histidine pathways. The molecular genetic characterization of purI mutants and their respective pseudorevertants resulted in the conclusion that <1% of the wild-type activity of the PurI enzyme was sufficient for thiamine but not for purine synthesis. The respective pseudorevertants were found to be informational suppressors. In addition, it was shown that accumulation of the purine intermediate aminoimidazole carboxamide ribotide inhibits thiamine synthesis, specifically affecting the conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine.

Aminoimidazole Carboxamide↗