Andrew L. Bognar

A domino effect in antifolate drug action in Escherichia coli

Mass spectrometry technologies for measurement of cellular metabolism are opening new avenues to explore drug activity. Trimethoprim is an antibiotic that inhibits bacterial dihydrofolate reductase (DHFR). Kinetic flux profiling with (15)N-labeled ammonia in Escherichia coli reveals that trimethoprim leads to blockade not only of DHFR but also of another critical enzyme of folate metabolism: folylpoly-gamma-glutamate synthetase (FP-gamma-GS). Inhibition of FP-gamma-GS is not directly due to trimethoprim. Instead, it arises from accumulation of DHFRs substrate dihydrofolate, which we show is a potent FP-gamma-GS inhibitor. Thus, owing to the inherent connectivity of the metabolic network, falling DHFR activity leads to falling FP-gamma-GS activity in a domino-like cascade. This cascade results in complex folate dynamics, and its incorporation in a computational model of folate metabolism recapitulates the dynamics observed experimentally. These results highlight the potential for quantitative analysis of cellular metabolism to reveal mechanisms of drug action.

Dissection of combinatorial control by the Met4 transcriptional complex.

Loss of Met31 and Met32 abolishes all Met4-activated transcription, while only certain target genes, such as sulfate assimilation genes, depend on Cbf1 and Met28 for expression. Unlike Met4 and the other cofactors, Cbf1 remains promoter-bound under inducing and repressing conditions and helps to stabilize Met32, the main platform for Met4, at promoters.

Cloning, and molecular characterization of the GCV1 gene encoding the glycine cleavage T-protein from Saccharomyces cerevisiae

We have isolated the gene encoding the glycine cleavage T-protein (GCV1) of the yeast Saccharomyces cerevisiae and shown through gene disruption and enzyme assays that inactivation of GCV1 destroys glycine cleavage function. A DNA fragment encoding the GCV1 gene was cloned by PCR amplification using degenerate oligodeoxyribonucleotides, and the cloned fragment was used as a probe to isolate the complete gene from a yeast genomic library. Growth with glycine stimulated expression of the GCV1 gene as determined by Northern analysis and increased the beta-galactosidase activity of a GCV1-lacZ fusion 30-fold. The URA3 gene was inserted into the coding sequence of GCV1 and the resulting construct was used to disrupt the chromosomal GCV1 gene in a diploid strain of yeast. gcv1::URA3 haploid derivatives grew normally or only slightly more slowly than the isogenic wild-type haploids. All gcv1 strains studied were unable to grow on glycine as a sole nitrogen source and lacked glycine cleavage enzyme activity. Growth of shm1 shm2 mutants was stimulated by glycine, whereas glycine could not supplement the growth of the isogenic gcv1 strain.

Transcriptional regulation of the one-carbon metabolism regulon in Saccharomyces cerevisiae by Bas1p.

The mechanisms mediating responses to glycine withdrawal in budding yeast were studied using a genome‐wide profiling approach. A striking pattern of repressed expression of genes with an enrichment for those involved in one‐carbon metabolism and AMP biosynthesis was revealed. Sequence analysis of the promoters for the most severely repressed genes identified a conserved sequence, TGACTC, a known binding site for the transcription factors Gcn4p and Bas1p. Loss of BAS1 abolished or significantly reduced the repression of these genes in response to glycine removal but this phenotype was much less apparent in the absence of BAS2 or GCN4. Addition of a Bas1p‐LexA fusion protein to a strain with a LexAop‐LacZ fusion showed a strong glycine effect both in a BAS2 and a bas2 background. A Bas1p‐VP16 fusion protein activated expression in a bas1bas2 strain but no glycine effect was observed while a Bas1p‐Bas2p fusion protein activated expression to a lesser extent with a slight stimulation by glycine. These results suggest that glycine affects Bas1p activation of transcription rather than DNA binding and that Bas2p is not required for this affect. Glycine withdrawal repressed many of the same genes as addition of adenine, a process known to be dependent on Bas1p. However, the glycine response is independent of adenine repression, because glycine regulation occurs normally in ade strains. We did not see any difference in the degree of stimulation by glycine in the presence or absence of adenine even in Ade+ strains. Glycine regulation was also found to be dependent on an intact SHM2 gene, which encodes cytoplasmic serine hydroxymethyltransferase. A reporter plasmid containing a DNA sequence from the GCV2 promoter which confers glycine regulation on heterologous genes was introduced into the yeast deletion set to screen for genes required for glycine regulation. A number of genes, including BAS1 were required for activation by glycine but only the SHM2 gene was required for repression in the absence of glycine. We also showed that regulation of the SHM2 promoter by glycine requires Bas1p but not Bas2p or Gcn4p using a β‐galactosidase reporter. The response of the promoter to glycine required an intact SHM2 gene but was restored in a shm2 strain by addition of formate to the medium.

Mutagenesis of Folylpolyglutamate Synthetase Indicates That Dihydropteroate and Tetrahydrofolate Bind to the Same Site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.

Mutation of Gly51 to serine in the P-loop of Lactobacillus casei folylpolyglutamate synthetase abolishes activity by altering the conformation of two adjacent loops.

Based upon the three-dimensional structure of Lactobacillus casei folylpolyglutamate synthetase (FPGS), site-directed mutagenesis studies were performed on three residues associated with the ATPase site: Gly51, Ser52 and Ser73. Gly51 and Ser52 are at the end of the P-loop, which is involved in triphosphate binding. A G51S mutant enzyme and a G51S/S52T double-mutant enzyme were made in order to alter the FPGS P-loop to more closely resemble the sequences found in other ATPase and GTPase enzymes. Ser73 is on a neighboring loop (the Omega-loop) and precedes a proline residue found to be in a cis conformation. The carbonyl O atom of Ser73 is one of the protein ligands for the essential Mg(2+) ion involved in ATP binding and hydrolysis and the Omega-loop is involved in binding the folate substrate 5,10-methylenetetrahydrofolate. The serine residue was mutated to alanine and this is the only one of the three mutants which retains some FPGS activity. The structures of the G51S, G51S/S52T and S73A mutant proteins have been solved to high resolution, along with the structure of the apo wild-type FPGS. The P-loop in both the G51S and G51S/S52T mutant proteins remains unaltered, yet both structures show a large conformational rearrangement of the Omega-loop in which a cis-Pro residue has switched conformation to a trans-peptide. The structure of the Omega-loop is severely disrupted and as a consequence structural rearrangements are observed in the peptide linker joining the two domains of the enzyme. Magnesium binding in the active site is also disrupted by the presence of the serine side chain at position 51 and by the repositioning of the carbonyl O atom of Ser73 and a water molecule is bound in place of the Mg(2+) ion. The S73A mutant protein retains the cis-Pro configuration in the Omega-loop and the Mg(2+) site remains intact. The cis-Pro is also observed in the structure of the substrate-free form of FPGS (apoFPGS), maintained in the absence of Mg(2+) by a hydrogen-bonding network involving water molecules in the active site. It is only in the complete absence of water or Mg(2+) in the binding site that the cis-Pro switches to the trans conformation.

Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding

Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS. Glu143 is positioned near the P-loop and interacts with the Mg(2+) of Mg NTP-binding proteins. We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+). The structure showed a water molecule at the place where Mg(2+) bound to the wild type enzyme. Mutant proteins E143A, and even E143D and E143Q with conservative mutations, lacked enzyme activity and failed to complement the methionine auxotrophy of the E. coli folC mutant SF4, showing that Glu143 is an essential residue. Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally. The E. coli E146Q mutant FPGS bound ADP with the same affinity as the wild type enzyme but bound ATP with much lower affinity and had higher ATPase activity than the wild type enzyme. The mutant enzyme was defective in forming the acyl-phosphate reaction intermediate from ATP and dihydropteroate. The E. coli FPGS requires activation by dihydropteroate or tetrahydrofolate binding to allow full activity. In the absence of a pteroate substrate, only 30% of the total enzyme binds ATP. We suggest that dihydropteroate causes a conformational change to allow increased ATP binding. The mutant enzyme was similarly activated by dihydropteroate resulting in increased ADP binding.

Molecular analysis of the folC gene of Pseudomonas aeruginosa.

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase‐dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co‐transcribed.

Purification and crystallization of Lactobacillus casei folylpolyglutamate synthetase expressed in Escherichia coli

Folylpolyglutamate synthetase (FPGS) from Lactobacillus casei has been crystallized with polyethylene glycol and acetate buffer at pH 5.0. The enzyme was obtained from Escherichia coli strain SF4 harboring the L. casei FPGS chromosomal gene on a pEMBL vector (pGT3-8.1). Crystals of the enzyme were obtained which diffract to 2.6 A resolution. The crystals are monoclinic, space group P2(1), with unit cell dimensions of a = 54.07 A, b = 45.83 A, c = 84.37 A and beta = 107.92 degrees. A unit cell contains one molecule of the 43,000 Da enzyme per asymmetric unit. A complete X-ray data set on the native crystals has been collected.

Microarray Analysis of Genes Induced by Methionine Starvation and Growth on Different Sulfur Sources in Yeast

Methionine is an important end-product of folate metabolism and it is reasonable to expect that the expression of several folate-dependent enzymes might be regulated by its availability. In the yeast, Saccharomyces cerevisiae, previous studies have shown that at least 20 genes of sulfate reduction and methionine biosynthesis are regulated by the transcription factor Met4p in response to methionine, cysteine or S-adenosylmethionine levels (1). Met4p lacks a DNA-binding domain and requires either Cbflp or the redundant factors Met31p or Met32p to bind to DNA at the promoters it regulates. Cbflp binds to the consensus sequence TCACGTG, while Met31 and Met32p both bind to the consensus sequence AAACTGTG. Another factor, Met28p increases the affinity of Cbflp binding and is also present in the Met4p-Met31p/Met32p promoter complex (2). Met4p is degraded or inhibited in response to excess methionine following ubiquitination by the SCFMET30complex (3, 4).