De novo purine nucleotide biosynthesis and regulation of the pathway in gram-positive bacteria


Purine nucleotides play an important role in many biochemical processes. ATP is the main source for energy. Adenine nucleotides serve as the components of coenzymes; Coenzyme A, FAD and NAD+. GTP is needed for activation of biochemical processes. In addition, the nucleotides serve as the building blocks of DNA and RNA. Cells synthesize purine nucleotides in two ways. The de novo synthesis proceeds via a 14-step pathway branching after IMP. The other way of synthesis is utilization of ready made purine bases in the salvage pathway. In these reactions, ribose phosphate is coupled to purine bases to produce IMP, AMP or GMP. Many purine nucleotide biosynthesis inhibitors are used therapeutically against cancer. Cancer cells divide faster than normal cells and thus require more purine nucleotides for DNA synthesis than normal cells. Disorders in the de novo purine nucleotide biosynthesis can cause gout. Except for viruses and certain parasites, all organisms can synthesize purine nucleotides de novo. The pathway is invariable, but the organization and regulation of the participating genes differ among organisms.

The genetic organization for the purine nucleotide biosynthesis is well known in Escherichia coli and Bacillus subtilis. In addition, genes for the de novo purine nucleotide biosynthesis have been cloned and characterized e.g. in Saccharomyces cerevisiae, Drosophila melanogaster, Dictyostelium discoideum, chicken, rat and human. The de novo biosynthesis is regulated at the gene level and at the enzyme level at least in E. coli, B. subtilis, S. cerevisiae and Lactococcus lactis. Glutamine 5-phosphoribosyl 1-pyrophosphate amidotransferase (glutamine PRPP amidotransferase) is the first enzyme in the biosynthetic pathway. Glutamine PRPP amidotransferases from S. cerevisiae, E. coli, B. subtilis and from mammalia are inhibited by AMP and GMP, the end products of the pathway. During normal growth conditions, purine nucleotide biosynthesis is regulated at enzyme level in E. coli. When excess of purine bases is available, E. coli controls the de novo pathway repressing the expression of the genes. The certain enzymes catalyzing de novo purine nucleotide biosynthesis in higher organisms are typically multifunctional.

De novo purine nucleotide biosynthesis in prokaryotes

The genes encoding the enzymes catalyzing purine nucleotide biosynthesis are scattered in S. cerevisiae, but constitute several monocistronic operons in E. coli. In B. subtilis, the genes encoding the enzymes catalyzing the ten first steps in the pathway are organized into a single operon. The genes form a regulon in E. coli and are regulated by PurR. Transcription of the pur operon in B. subtilis is repressed by PurR in the presence of adenine and separately regulated by attenuation.

Purine nucleotide biosynthesis in Bacillus subtilis

The genes in de novo synthesis of IMP constitute an operon in B. subtilis. This pur operon contains 12 genes at 55° on the genetic map. The gene and enzyme ratio is the same in E. coli and B. subtilis except that in B. subtilis two genes, purQ and purL, are needed to encode one enzyme, FGAM synthetase. The purQ gene encodes a glutamine dependent amidotransferase subunit and purL encodes an ammonia dependent amidotransferase subunit. The pur operon contains three clusters of overlapping genes separated by intercistronic spaces: purEKB-(73 bp)-purCSQLF-(101 bp)-purMNH(J)-(15 bp)purD. The 84-codon purS is required for coupling the subunits of FGAM synthetase, and purH(J) is a gene couple encoding a bifunctional enzyme that catalyzes steps 9 and 10 of the de novo biosynthesis of purine nucleotides. Transcription of the pur operon is initiated 242 bp upstream from the purE start codon. Four nucleotides upstream from the transcription initiation is a putative s43promoter site, TTGACA-(N17)-TAAGAT. The existence of polycistronic operons is typical for the de novo biosynthetic genes in B. subtilis. The other well-characterized gene sets are in the pathways for pyrimidine nucleotide, tryptophan and arginine syntheses.

Adenylosuccinate synthetase (sAMP synthetase), one of the two enzymes catalyzing the synthesis of AMP is encoded by a single gene operon, purA operon. Transcription of the gene starts 44 bp upstream from the initiation codon of purA. In the front of transcription initiation site there is a putative s43promoter element, TTGACT-(N17)-TAAACT. The two enzymes, GMP synthetase and IMP dehydrogenase catalyze the synthesis of GMP from IMP. The enzymes are encoded by two genes, guaA and guaB, respectively. The two genes function as separate operons. Transcription of the guaA operon initiates at a s43 promoter, TTGACC-(N17)-TAGAAT. The guaA transcript contains a 130 nucleotides long nontranslated leader sequence. There is no experimental evidence for the initiation site of guaB transcription. However, a putative promoter element, TTGACA-(N l g)TAATCT is located at 38 bp upstream from the translation initiation codon.

Regulation of de novo purine nucleotide biosynthesis in B. subtilis

The pur operon is primarily regulated by repression and attenuation. However, the translational regulation of the operon is not excluded. Regulation of purA, guaA and guaB operons is not clear, but the available data suggest close similarity to the regulation of the pur operon. Adenine and guanine are known to decrease synthesis of glutamine PRPP amidotransferase. Adenine prevents initiation of transcription and guanine promotes termination of transcription at the 242 bp long leader sequence. The effect of purines is cumulative and de novo purine nucleotide synthesis is almost completely prevented by simultaneous addition of adenine and guanine.

In the presence of guanine, cells tend to accumulate appr. 200 nucleotides long mRNA and the amount of full-length mRNA decreases. The 242 bp long nontranslated leader sequence contains a potential secondary structure typical for factor-independent transcription termination. This putative terminator could be part of a mechanism for guanine-mediated regulation of transcription. There is an additional potential overlapping secondary structure that could function in antitermination by precluding the formation of the terminator. In the pur operon, a regulatory protein may be activated by guanine compounds and by binding to the leader sequence may destroy and/or prevent the formation of the antitermination loop structure and allow the formation of the termination loop. When guanine pool is insufficient to activate the regulatory protein, the anti-termination loop effectively prevents the formation of premature transcripts.

Addition of adenine represses transcription initiation of the full length and premature mRNA of the pur operon indicating that adenine-mediated repression is an independent control mechanism by which the cells regulate de novo purine nucleotide synthesis. The repression by adenine is mediated by purine repressor, PurR. The binding of PurR to the control region of the pur operon is inhibited by PRPP. Thus, the intracellular level of PRPP regulates the transcription of the pur operon. Uptake of adenine decreases the PRPP pool in the cell leading to repression of the pur operon.

Excess of adenine in the culture medium decreases synthesis of sAMP synthetase. Adenine nucleotides may prevent the initiation of purA transcription analogously to pur operon. Guanine is not inhibitory to purA expression. Guanosine and especially GMP, on the other hand, decrease guaB expression. Regulation of guaA is not clear. Addition of guanosine, xanthine, hypoxanthine or guanosine and adenine into minimal growth medium has no effect on synthesis of GMP synthetase. However, guaA transcript has a 130 nucleotides long nontranslated leader sequence which by analogy to pur operon might form mutually exclusive antitermination and termination secondary structures. The putative termination loop is followed by a purine rich region typical to the r -independent termination structure. However, the exact role of the leader sequence is not yet clear.

The aim of present study

Present work will focus on purine nucleotide biosynthesis in gram-positive organisms B. subtilis, Lactic acid bacteria and Streptomyces species are important industrial microorganisms because of their role in food fermentations and antibiotic production. Lactococci are widely used in dairy fermentations as a starter culture to produce variety of cheeses and fermented milk products. Most strains of Lactococci carry plasmids of a variety of functional properties i.e. lactose metabolism, proteinase activity, nisin production and resistance.

Despite the fact that the genes for de novo purine nucleotide biosynthesis have been isolated from a number of organisms, only in E. coli and B. subtilis the enzymes and regulation of the biosynthetic genes have been characterized in more detail. In bacteria, expression of each of the genes is regulated, but in animals there is no evidence yet for gene regulation in this pathway. In E. coli the pur loci responsible for de novo purine biosynthesis consists of scattered operons and a single locus, but in B. subtilis all of the genes needed for biosynthesis of IMP, the precursor of the purine nucleotides are in one, purEKBCSQLMNH(J)D operon. No genes of the purine nucleotide biosynthesis have yet been cloned in L. lactis and Streptomyces spp.

The first enzyme of the biosynthesis pathway, glutamine PRPP amidotransferase is likely to be the key enzyme in the regulation of the pathway. Actually, the enzyme has been shown to be itself subject to multiple controls in E. coli and B. subtilis. The sequence homology of the known glutamine PRPP amidotransferases is relatively high. Nevertheless, the enzymes exhibit major structural differences, E. coli and B. subtilis enzymes represent two different types of glutamine PRPP amidotransferases. The B. subtilis enzyme has an Fe-S cluster essential for activity and a propeptide, which needs to be processed to exposure active site cysteine, Cys-1, while E. coli enzyme has no Fe-S-cluster and no propeptide.

Purine nucleotide biosynthesis is regulated by two mechanisms in B. subtilis. Guanine addition causes premature transcription of the pur operon, whereas addition of adenine leads to the PurR-mediated repression of transcription initiation. Present work will focus on purine nucleotide biosynthesis in gram-positive organisms B. subtilis. In bacteria, expression of each of the genes is regulated, but in animals there is no evidence yet for gene regulation in this pathway. In E. coli the pur loci responsible for de novo purine biosynthesis consists of scattered operons and a single locus, but in B. subtilis all of the genes needed for biosynthesis of IMP, the precursor of the purine nucleotides are in one, purEKBCSQLMNH(J)D operon..

Earlier it has been reported that adenine decreases sAMP synthetase production in B. subtilis and guanosine increases the synthesis of sAMP synthetase. However, the studies were obtained by measuring the enzyme levels in different growth conditions. It is clear that more precise analysis is needed. One of our aims is to study regulation of purA in more detail. Given that PurR mediates the repression of purA analogously to the pur operon, the repression mechanism of PurR can be studied more accurately than by using pur operon, in which the other regulation mechanism, transcriptional attenuation, interferes with the repression. The organization and regulation of the genes in different gram-positive bacteria and the structure and function of the components participating in regulation of purine nucleotide pathway is another important aspect we are interested in. Our aim is to study in details the control mechanisms and to compare the mechanisms in different gram-positive bacteria.