Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5''-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis.

It has been shown that the Escherichia coli purE locus specifying 5'-phosphoribosyl-5-amino-4-imidazole carboxylase in de novo purine nucleotide synthesis is divided into two cistrons. We cloned and determined a 2,449-nucleotide sequence including the purE locus. This sequence contains two overlapped open reading frames, ORF-18 and ORF-39, encoding proteins with molecular weights of 18,000 and 39,000, respectively. The purE mutations of CSH57A and DCSP22 were complemented by plasmids carrying ORF-18, while that of NK6051 was complemented by plasmids carrying ORF-39. Thus, the purE locus consists of two distinct genes, designated purE and purK for ORF-18 and ORF-39, respectively. These genes constitute a single operon. A highly conserved 16-nucleotide sequence, termed the PUR box, was found in the upstream region of purE by comparing the sequences of the purF and purMN operons. We also found three entire and one partial repetitive extragenic palindromic (REP) sequences in the downstream region of purK. Roles of the PUR box and REP sequences are discussed in relation to the genesis of the purEK operon.     

Genomic organization of purK and purE in Brevibacterium ammoniagenes ATCC 6872: purE locus provides a clue for genomic evolution

Abstract From the genomic library of Brevibacterium ammoniagenes ATCC6872, the purE locus encoding 5'-phosphoribosyl-5aminoimidazole (AIR) carboxylase (EC 4.1.1.21) was cloned and its nucleotide sequence was determined. From the sequence analysis, two distinct open reading frames (ORFs) in the sequence of the purE locus were identified as purK and purE genes ( purK-purE ). An in vivo translation experiment reconfirmed the purK and purE genes to be independent. The genomic organization in the purE locus of B. ammoniagenes is opposite to that of the bacteria Escherichia coli and Bacillus subtilis . However, it coincides with the fused genes ( purKE ) of higher organisms Saccharomyces cerevisiae, Schizosaccharomyces pombe and Vigna aconitifolia . This suggests that the purE locus might be an intermediate form for genomic evolution of bacteria to higher organisms.     

Identification and nucleotide sequence of a gene encoding 5'-phosphoribosylglycinamide transformylase in Escherichia coli K12

5'-Phosphoribosylglycinamide transformylase (EC 2.1.2.2), encoded by the purN gene of Escherichia coli, catalyzes the synthesis of 5'-phosphoribosylformylglycinamide from 5'-phosphoribosylglycinamide (GAR). The mature protein, as deduced from the purN structural gene sequence, contains 212 amino acid residues and has a calculated Mr of 23,241. The purN gene is located adjacent to and immediately downstream from the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase where the initiation codon for GAR transformylase overlaps the termination codon of AIR synthetase. Based on polarity studies, the expression of the purN gene originates from the purM control region and thus forms a purMN operon. The E. coli GAR transformylase shows greater homology to the GAR transformylase domain of the trifunctional Gart polypeptide of Drosophila than to the single GAR transformylase of Saccharomyces. Immediately downstream from the purN gene of the purMN operon is a region of dyad symmetry capable of forming a hairpin stem and loop structure characteristic of a rho-independent terminator.     

Treponema denticola PurE Is a bacterial AIR carboxylase

De novo purine biosynthesis proceeds by two divergent paths. In bacteria, yeasts, and plants, 5-aminoimidazole ribonucleotide (AIR) is converted to 4-carboxy-AIR (CAIR) by two enzymes: N(5)-carboxy-AIR (N(5)-CAIR) synthetase (PurK) and N(5)-CAIR mutase (class I PurE). In animals, the conversion of AIR to CAIR requires a single enzyme, AIR carboxylase (class II PurE). The CAIR carboxylate derives from bicarbonate or CO(2), respectively. Class I PurE is a promising antimicrobial target. Class I and class II PurEs are mechanistically related but bind different substrates. The spirochete dental pathogen Treponema denticola lacks a purK gene and contains a class II purE gene, the hallmarks of CO(2)-dependent CAIR synthesis. We demonstrate that T. denticola PurE (TdPurE) is AIR carboxylase, the first example of a prokaryotic class II PurE. Steady-state and pre-steady-state experiments show that TdPurE binds AIR and CO(2) but not N(5)-CAIR. Crystal structures of TdPurE alone and in complex with AIR show a conformational change in the key active site His40 residue that is not observed for class I PurEs. A contact between the AIR phosphate and a differentially conserved residue (TdPurE Lys41) enforces different AIR conformations in each PurE class. As a consequence, the TdPurE·AIR complex contains a portal that appears to allow the CO(2) substrate to enter the active site. In the human pathogen T. denticola, purine biosynthesis should depend on available CO(2) levels. Because spirochetes lack carbonic anhydrase, the corresponding reduction in bicarbonate demand may confer a selective advantage.     

Genetic Analysis of Metabolic Crosstalk and Its Impact on Thiamine Synthesis in Salmonella Typhimurium

The first five steps in de novo purine biosynthesis are involved in the formation of the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine. We show here that the first enzyme in de novo purine biosynthesis, PurF, is required for thiamine synthesis during aerobic growth on some but not other carbon sources. We show that PurF-independent thiamine synthesis depends on the recently described alternative pyrimidine biosynthetic (APB) pathway. Null mutations in zwf (encoding glucose-6-P dehydogenase), gnd (encoding gluconate-6-P dehydrogenase), purE (encoding aminoimidazole ribo-nucleotide carboxylase), and purR (encoding a regulator of gene expression) were found to affect the function of the APB pathway. A model is presented to account for the involvement of these gene products in thiamine biosynthesis via the APB pathway. Results presented herein demonstrate that function of the APB pathway can be prevented either by blocking intermediate formation or by diverting intermediate(s) from the pathway. Strong genetic evidence supports the conclusion that aminoimidazole ribotide (AIR) is an intermediate in the APB pathway.     

Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway.

Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity. PurE separates from PurK in the first ammonium sulfate fractionation during the purification. No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained. To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity. The activities of PurE, PurK, and PurE.PurK have been investigated. PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate. The Km for HCO3- in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR. No bicarbonate dependence on this reaction could be demonstrated (less than 100 microM), and AIR is not carboxylated during the hydrolysis of ATP. Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (less than 100 microM) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and Pi. No dependence on the concentration of HCO3- could be demonstrated. A new energy requirement in the purine biosynthetic pathway has been established.     

Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus

The de novo purine biosynthetic enzymes 5-amino-4-imidazolecarboxamide-ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide-ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli. The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr 57,335 and 45,945 (GAR synthetase), respectively, that formed an operon. The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57,335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR transformylase and IMP cyclohydrolase activities. The 5' end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming 'hairpin' loops where the formation of the one would prevent the formation of the other but not vice versa. Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.     

Nucleotide sequence and analysis of the purA gene encoding adenylosuccinate synthetase of Escherichia coli K12

Adenylosuccinate synthetase (EC 6.3.4.4), encoded by the purA gene of Escherichia coli K12, catalyzes the synthesis of adenylosuccinate (SAMP) from IMP, the first committed step in AMP biosynthesis. The E. coli K12 purA gene and flanking DNA was cloned by miniMu-mediated transduction, and the nucleotide sequence was determined. The mature SAMP synthetase subunit, as deduced from the DNA sequence, contains 427 amino acid residues and has a calculated Mr of 47,277. The size of the purA mRNA was determined by Northern blotting to be approximately 1.5 kilobase pairs. The 5'-end of the purA mRNA was identified by primer extension and is located 23 nucleotides upstream of the ATG translational initiation codon. Comparison of the purA control region with the guaBA control region revealed a common region of dyad symmetry which may suggest mutual elements of regulation. The purA control region did not resemble the control regions of the other known pur loci.     

Nucleotide sequence of cDNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase from maize

We have cloned a full length cDNA for the small subunit of ribulose-1,5-bisphosphate carboxylase from C4 monocot maize, determined the complete nucleotide sequence of this cDNA and deduced its amino acid sequence. The cDNA insert included 513 bp of the coding region, and 65 and 252 nucleotides of the 5' and 3' untranslated regions, respectively. The transit and mature peptides have, respectively, 47 and 123 amino acids. Comparison with the small subunit genes from other plants revealed that the maize small subunit is similar to the wheat one, there being 73% homology between the transit peptides and 64% between the mature proteins. This indicates that there is no noteworthy difference between the C3 and C4 small subunit structures. Extreme codon bias was observed for this gene, and similar codon preferences are observed for other proteins highly expressed in maize leaf, light harvesting chlorophyll binding protein and phosphoenolpyruvate carboxylase. The results indicate that preferential codon usage for highly expressed genes occurs in maize leaf.     

The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase

The gene for the large subunit (LS) of ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBPCase/ Oase) from tobacco has been cloned in pBR322 and sequenced. The coding region contains 1431 bp (477 codons). The deduced arnino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS. The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6–10 bp upstream from the initiation codon.     

Structural and functional relationships between fumarase and aspartase. Nucleotide sequences of the fumarase (fumC) and aspartase (aspA) genes of Escherichia coli K12.

The nucleotide sequences of two segments of DNA (2250 and 2921 base-pairs) containing the functionally related fumarase (fumC) and aspartase (aspA) genes of Escherichia coli K12 were determined. The fumC structural gene comprises 1398 base-pairs (466 codons, excluding the initiation codon), and it encodes a polypeptide of Mr 50353 that resembles the fumarases of Bacillus subtilis 168 (citG-gene product), rat liver and pig heart. The fumC gene starts 140 base-pairs downstream of the structurally-unrelated fumA gene, but there is no evidence that both genes form part of the same operon. The aspA structural gene comprises 1431 base-pairs (477 codons excluding the initiation codon), and it encodes a polypeptide of Mr 52190, similar to that predicted from maxicell studies and for the enzyme from E. coli W. Remarkable homologies were found between the primary structures of the fumarase (fumC and citG) and aspartase (aspA) genes and their products, suggesting close structural and evolutionary relationships.     

The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase.

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.     

Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction.

Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP. The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains. These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon. Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR. Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression.     

Nucleotide sequence of the 5'' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5'' splicing site for subgenomic mRNAs

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).