Reduced flux through the purine biosynthetic pathway results in an increased requirement for coenzyme A in thiamine synthesis in Salmonella enterica serovar typhimurium

Work presented here establishes a connection between cellular coenzyme A (CoA) levels and thiamine biosynthesis in Salmonella enterica serovar Typhimurium. Prior work showed that panE mutants (panE encodes ketopantoate reductase) had a conditional requirement for thiamine or pantothenate. Data presented herein show that the nutritional requirement of panE mutants for either thiamine or pantothenate is manifest only when flux through the purine biosynthetic pathway is reduced. Further, the data show that under the above conditions it is the lack of thiamine pyrophosphate, and not decreased CoA levels, that directly prevents growth.     

Betaine-Homocysteine Transmethylase in Pseudomonas denitrificans, a Vitamin B12 Overproducer

A pantothenate-methionine auxotroph (J741) of Pseudomonas denitrificans was isolated whose growth requirement for methionine could not be satisfied by known precursors of the amino acid, including homocysteine. However, some "methyl rich" compounds such as betaine and dimethylacetothetin (DMT) could satisfy the requirement. S-Methyl-methionine and S-adenosylmethionine were ineffective. Extracts were found to contain an enzyme, betaine-homocysteine transmethylase (BHTase), that uses betaine or DMT as a methyl donor and homocysteine as an acceptor to produce methionine. Growth of J741 in methionine leads to a total repression of the BHTase, whereas the use of DMT leads to a three- to sixfold stimulation of enzyme synthesis compared to betaine-grown cells. The pantothenate requirement is unrelated to the methionine auxotrophy, since the growth of other single auxotrophic mutants as well as revertants of J741 still have their methionine requirement satisfied by betaine or DMT. Another methionine auxotroph that could not use betaine for growth was devoid of BHTase activity.     

Involvement of the oxidative pentose phosphate pathway in thiamine biosynthesis in Salmonella typhimurium.

purF mutants of Salmonella typhimurium are known to require a source of both purine and thiamine; however, exogenous pantothenate may be substituted for the thiamine requirement. We show here that the effect of pantothenate is prevented by blocks in the oxidative pentose phosphate pathway, gnd (encoding gluconate 6-phosphate [6-P] dehydrogenase) or zwf (encoding glucose 6-P dehydrogenase). We further show that the defects caused by these mutations can be overcome by increasing ribose 5-P, suggesting that ribose 5-P may play a role in the ability of pantothenate to substitute for thiamine.     

Mechanism of adenine inhibition in adenine-sensitive mutants of Salmonella typhimurium

Dalal, Fram R. (University of Pennsylvania, Philadelphia), Ronald E. Gots, and Joseph S. Gots. Mechanism of adenine inhibition in adenine-sensitive mutants of Salmonella typhimurium. J. Bacteriol. 91: 507-513. 1966.-The inhibition of growth of Salmonella typhimurium by adenine was studied with three adenine-sensitive mutants. These mutants were acutely sensitive to inhibition by adenine, were prototrophic in their growth requirements, and represented mutational events in three different genetic loci. In all cases, inhibition by adenine was relieved noncompetitively by thiamine (or its pyrimidine moiety), pantothenate (or its pantoyl moiety), and methionine alone or, more efficiently, in the presence of lysine. Kinetics of reversal indicated that adenine inhibited the synthesis of the reversing agents, probably at the level of a common factor required for their syntheses, such as the folic acid coenzymes. Support for this inference has been found by the facts that one of the mutants was identified as a partial auxotroph for p-aminobenzoic acid, and sulfadiazine could sensitize the wild type to acute inhibition by adenine.     

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.     

Lesions in gshA (Encoding gamma-L-glutamyl-L-cysteine synthetase) prevent aerobic synthesis of thiamine in Salmonella enterica serovar typhimurium LT2

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.     

Altered pathway routing in a class of Salmonella enterica serovar Typhimurium mutants defective in aminoimidazole ribonucleotide synthetase

In Salmonella enterica serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the purI gene in S. enterica. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways, purI mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37-44, 1996). Unexpectedly, some mutant purI alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The K(m) of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the V(max) was decreased 2.5-fold. The K(m) for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and purR null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.     

Pantothenate and coenzyme A in bacterial growth

Toennies, G. (Temple University School of Medicine, Philadelphia, Pa.), D. N. Das, and F. Feng. Pantothenate and coenzyme A in bacterial growth. J. Bacteriol. 92:707-713. 1966.-The effect of environmental pantothenate levels on the growth of Streptococcus faecalis 9790 was studied in terms of growth rate, depletion phenomena, cellular coenzyme A (CoA) content, and differential rates of wall and membrane synthesis. Low concentrations of pantothenate yielded normal exponential growth curves up to peak turbidities which are a function of pantothenate concentration. Attainment of these peaks was followed by lysis. Under such conditions, bacterial CoA increased initially in proportion with cell substance, but attained a peak level much earlier than cell substance, and then gradually decreased down to vanishing amounts. With higher pantothenate concentrations, cellular CoA levels increased to a maximum, and, under these conditions, the CoA content remained constant during exponential growth. Four-fifths of the pantothenate requirement of growing cells was eliminated by environmental oleate and palmitate. When CoA disappeared during growth on low pantothenate levels, cell wall synthesis seemed to continue at nearly normal rates, but membrane synthesis was severely curtailed. The data suggest that in fermentative organisms pantothenate action might be confined to wall and membrane synthesis, that these two processes differ in their quantitative dependence on pantothenate, and that pantothenate might occur in the form of acyl carrier protein as well as CoA.     

Identification of the heme axial ligands in the cytochrome b562 of the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (SDH) plays a key role in energy generation by coupling the oxidation of succinate to the reduction of ubiquinone in the mitochondrial electron transport chain. The Saccharomyces cerevisiae SDH is composed of a catalytic dimer of the Sdh1p and Sdh2p subunits containing flavin adenine dinucleotide (FAD) and iron-sulfur clusters and a heme b-containing membrane-anchoring domain comprised of the Sdh3p and Sdh4p subunits. We systematically mutated all the histidine and cysteine residues in Sdh3p and Sdh4p to identify the residues involved in axial heme ligation. The mutants were characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction, for heme b content, and for heme spectral properties. Mutation of Sdh3p His-46 or His-113 leads to a marked reduction in the catalytic efficiency of the enzyme for quinone reduction, suggesting that these residues form part of a quinone-binding site. We identified Sdh3p His-106 and Sdh4p Cys-78 as the most probable axial ligands for cytochrome b(562). Replacement of His-106 or Cys-78 with an alanine residue leads to a marked reduction in cytochrome b(562) content and to altered heme spectral characteristics that are consistent with a direct perturbation of heme b environment. This is the first identification of a cysteine residue serving as an axial ligand for heme b in the SDH family of enzymes. Loss of cytochrome b(562) has no effect on enzyme assembly and quinone reduction; the role of the heme in enzyme structure and function is discussed.     

Evidence for a new, oxygen-regulated biosynthetic pathway for the pyrimidine moiety of thiamine in Salmonella typhimurium.

The synthesis of the pyrimidine moiety of thiamine (vitamin B1) shares five reactions with the de novo purine biosynthetic pathway. Aminoimidazole ribotide (AIR) is the last common intermediate before the two pathways diverge. Evidence for the existence of a new pathway to the pyrimidine which bypasses the de novo purine biosynthetic pathway is reported here. This pathway is only expressed under anaerobic growth conditions and is denoted alternative pyrimidine biosynthesis or APB. Labeling studies are consistent with pantothenate being a precursor to the pyrimidine moiety of thiamine that is synthesized by the APB pathway. The APB pathway is independent of the alternative purF function which was proposed previously (D. M. Downs and J. R. Roth, J. Bacteriol. 173:6597-6604, 1991). The alternative purF function is shown here to be affected by temperature and exogenous pantothenate. Although the evidence suggests that the APB pathway is separate from the alternative purF function, the relationship between this function and the APB pathway is not yet clear.     

1-methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism

In Salmonella enterica serovar Typhimurium a mutation in the purF gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a purF mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from S. enterica specific for leucine, proline, and arginine contain 1-methylguanosine (m(1)G37) adjacent to and 3' of the anticodon (position 37). The formation of m(1)G37 is catalyzed by the enzyme tRNA(m(1)G37)methyltransferase, which is encoded by the trmD gene. Mutations in this gene, which result in an m(1)G37 deficiency in the tRNA, in a purF mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m(1)G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m(1)G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.     

Defects in pyruvate kinase cause a conditional increase of thiamine synthesis in Salmonella typhimurium.

As genomic sequence data become more prevalent, the challenges in microbial physiology shift from identifying biochemical pathways to understanding the interactions that occur between them to create a robust but responsive metabolism. One of the most powerful methods to identify such interactions is in vivo phenotypic analysis. We have utilized thiamine synthesis as a model to detect subtle metabolic interactions due to the sensitivity allowed by the small cellular requirement for this vitamin. Although purine biosynthesis produces an intermediate in thiamine synthesis, mutants blocked in the first step of de novo purine biosynthesis (PurF) are able to grow in the absence of thiamine owing to an alternative synthesis. A number of general metabolic defects have been found to prevent PurF-independent thiamine synthesis. Here we report stimulation of thiamine-independent growth caused by a mutation in one or both genes encoding the pyruvate kinase isozymes. The results presented herein represent the first phenotype described for mutants defective in pykA or pykF, and thus identify metabolic interactions that exist in vivo.     

Use of pantothenate as a metabolic switch increases the genetic stability of farnesene producing Saccharomyces cerevisiae

We observed that removing pantothenate (vitamin B₅), a precursor to co-enzyme A, from the growth medium of Saccharomyces cerevisiae engineered to produce β-farnesene reduced the strain׳s farnesene flux by 70%, but increased its viability, growth rate and biomass yield. Conversely, the growth rate and biomass yield of wild-type yeast were reduced. Cultivation in media lacking pantothenate eliminates the growth advantage of low-producing mutants, leading to improved production upon scale-up to lab-scale bioreactor testing. An omics investigation revealed that when exogenous pantothenate levels are limited, acyl-CoA metabolites decrease, β-oxidation decreases from unexpectedly high levels in the farnesene producer, and sterol and fatty acid synthesis likely limits the growth rate of the wild-type strain. Thus pantothenate supplementation can be utilized as a “metabolic switch” for tuning the synthesis rates of molecules relying on CoA intermediates and aid the economic scale-up of strains producing acyl-CoA derived molecules to manufacturing facilities.     

The Saccharomyces cerevisiae succinate dehydrogenase anchor subunit, Sdh4p: mutations at the C-terminal lys-132 perturb the hydrophobic domain

The yeast succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits, Sdh1p-Sdh4p, that couples the oxidation of succinate to the transfer of electrons to ubiquinone. One of the membrane anchor subunits, Sdh4p, has an unusual 30 amino acid extension at the C-terminus that is not present in SDH anchor subunits of other organisms. We identify Lys-132 in the Sdh4p C-terminal region as necessary for enzyme stability, ubiquinone reduction, and cytochrome b562 assembly in SDH. Five Lys-132 substituted SDH4 genes were constructed by site-directed mutagenesis and introduced into an SDH4 knockout strain. The mutants, K132E, K132G, K132Q, K132R, and K132V were characterized in vivo for respiratory growth and in vitro for ubiquinone reduction, enzyme stability, and cytochrome b562 assembly. Only the K132R substitution, which conserves the positive charge of Lys-132, produces a wild-type enzyme. The remaining four mutants do not affect the ability of SDH to oxidize succinate in the presence of the artificial electron acceptor, phenazine methosulfate, but impair quinone reductase activity, enzyme stability, and heme insertion. Our results suggest that the presence of a positive charge on residue 132 in the C-terminus of Sdh4p is critical for establishing a stable conformation in the SDH hydrophobic domain that is compatible with ubiquinone reduction and cytochrome b562 assembly. In addition, our data suggest that heme does not play an essential role in quinone reduction.     

Flavinylation and Assembly of Succinate Dehydrogenase Are Dependent on the C-terminal Tail of the Flavoprotein Subunit

The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ∼70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg⁵⁸² in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg⁶³⁸ compromises SDH function only when present in combination with a Cys⁶³⁰ substitution. Mutations of either Arg⁵⁸² or Arg⁶³⁸/Cys⁶³⁰ do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits.     

Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p paralogs results in catalytically active succinate dehydrogenase isoenzymes

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.