Novel deoxynucleoside‐phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides

Wang, Liya; Westberg, Joakim; Bölske, Göran; Eriksson, Staffan

doi:10.1046/j.1365-2958.2001.02700.x

Cited by 43 publications

(62 citation statements)

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“…Mycoplasma species are thought to be completely dependent on their host for supplying these nucleic acid precursors. Genomic sequence analyses of a number of Mycoplasma species have identified novel enzymes associated with salvage pathways for biosynthesis of nucleic acids from nucleotides (41,53,54). Mycoplasma nucleases are thought to play a metabolic role in the production of nucleotide substrates from host or microbial nucleic acids released through natural and induced cell death.…”

Section: Discussionmentioning

confidence: 99%

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Wakamatsu

Kim

Uemura

et al. 2011

Journal of Biological Chemistry

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RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3-phosphoadenosine 5-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide-and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5 to 3, in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5-3 direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.Nucleases are essential for nucleic acid metabolism and cell viability. DNA repair and recombination require a variety of specific deoxyribonucleases (1, 2). RNA metabolism also requires various ribonucleases (RNases) 2 for a broad range of processes (3, 4). In mRNA turnover, mRNA decay is an important determinant of gene expression and is mediated by several RNases with endonuclease and exonuclease activity. Nucleases belonging to different superfamilies have been identified with endonuclease and exonuclease activities (5-8).In addition, genome sequences have revealed numerous ORFs annotated as putative nucleases.One of the phosphoesterase families is the DHH superfamily (9), which includes phosphodiesterase and exopolyphosphatase (10 -12). RecJ is a DNA exonuclease in the DHH protein superfamily, with DHH motifs I-IV and an associated DHHA1 motif (9), as described in the Pfam database (13). The RecJ protein is a Mg 2ϩ -or Mn 2ϩ -dependent single-stranded DNA (ssDNA)-specific 5Ј-3Ј exonuclease/deoxyribophosphodiesterase and plays a role in homologous recombination, mismatch repair, and base excision repair (14 -19). Although the ORFs annotated as RecJ-related proteins also have DHH and DHHA1 motifs, each RecJ-related protein has to be investigated for nuclease activity similar to RecJ protein. According to the SYSTERS database (20), proteins with DHH and DHHA1 motifs can be further divided into three main families. Representatives of each family are RecJ (N_155469 in SYSTERS), putative poly(A) polymerase (O_142140), and ex...

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Section: Discussionmentioning

confidence: 99%

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Wakamatsu

Kim

Uemura

et al. 2011

Journal of Biological Chemistry

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“…mycoides (Rodwell, 1960) and Acholeplasma laidlawii (Rodwell and Mitchell, 1979), which became biochemical research models for Mollicutes (Pollack et al, 1997). These studies revealed the general metabolic capabilities of these organisms and some exciting new findings, such as PPi-dependent nucleoside kinase activities, never described previously in any living organism (Tryon and Pollack, 1984;Wang et al, 2001).…”

Section: Introductionmentioning

confidence: 85%

“…When converting nucleobases into nucleosides, these enzymes can accept a deoxyribose-1-phosphate instead of ribose-1-phosphate, generating deoxyribonucleosides. It was also found that mycoplasmas have the ability to phosphorylate all natural deoxynucleosides via deoxynucleoside kinases with ATP-dependent activities but also, in some species, NMP-dependent (phosphotransferases) and PPi-dependent activities (Wang et al, 2001).…”

Section: Purine and Pyrimidine Nucleotide Pathways In Mollicutesmentioning

confidence: 99%

Purine and pyrimidine nucleotide metabolism in Mollicutes

Bizarro

Schuck

2007

Genet. Mol. Biol.

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Several mollicute genome projects are underway, offering unique opportunities to study genes and metabolic pathways on a genome-wide scale. Here, we have analyzed the conservation and diversity of purine and pyrimidine metabolism in mycoplasmas. An evaluation of discrepancies between genomic analysis and enzymatic data revealed interesting aspects about these organisms. We found important examples in which enzyme activity was reported without the annotation of a corresponding gene. An interesting example concerns phosphopentomutase. In Mollicutes, we have identified CDSs orthologous to sequences recently identified as new phosphopentomutases in archaeobacteria that are structurally related to phosphomannomutases. It is suggested that these sequences could replace the function of phosphopentomutases in mollicutes lacking the canonical phosphopentomutase gene (deoB). Also, the activity of 5'-nucleotidase was reported in mollicutes that do not possess any CDS related to ushA. Hypothetical proteins exhibiting domains similar to newly characterized 5' nucleotidases in Escherichia coli are proposed as possible CDSs related to this enzymatic activity in Mollicutes. Based on our analysis, the reductive genome evolution of Mollicutes does not appear to result in a minimum set of genes nor a minimum set of metabolic functions shared by all mollicute species.

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“…However, genomic sequence analyses of a number of mycoplasma species have identified novel enzymes associated with salvage pathways for the biosynthesis of nucleic acids from nucleotides (5,21,24,34,42,44,51,(57)(58)(59). These salvage pathways have been functionally characterized in Mycoplasma mycoides subspecies mycoides (35)(36)(37)58). Mycoplasma nucleases are thought to play a metabolic role in the production of nucleotide substrates from host or microbial nucleic acids released through natural and induced cell death.…”

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confidence: 99%

Mycoplasma hyopneumoniae mhp379 Is a Ca ²⁺ -Dependent, Sugar-Nonspecific Exonuclease Exposed on the Cell Surface

2007

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Mycoplasma hyopneumoniae mhp379 is a putative lipoprotein that shares significant amino acid sequence similarity with a family of bacterial thermostable nucleases. To examine the nuclease activity of mhp379, the gene was cloned and expressed in Escherichia coli following the deletion of the amino-terminal signal sequence and prokaryotic lipoprotein cleavage site and mutagenesis of the mycoplasma TGA tryptophan codons to TGG. The recombinant fusion protein yielded a 33-kDa thrombin cleavage product, corresponding in size to the mature mhp379 protein. Exonuclease activity was indicated by agarose gel electrophoresis analysis of the reaction products that were released when different nucleic acid substrates were used. Endonuclease activity was also indicated by the digestion of closed circular plasmid DNA. The recombinant mhp379 fusion protein completely digested single-stranded DNA, double-stranded DNA (dsDNA), and RNA. The optimal reaction conditions were determined with a novel nuclease assay based on the enhancement of fluorescence of SYBR green I bound to dsDNA. Optimal activity was observed in the presence of calcium ions at a concentration of 15 mM and a pH of 9.5. No nuclease activity was observed in the absence of calcium ions. Mycoplasmas do not have the ability to synthesize nucleic acid precursors, and thus, nucleases are likely to be important in the acquisition of precursors for the synthesis of nucleic acids. Homologs of an ATP-binding cassette (ABC) transport system were identified immediately downstream of the gene encoding mhp379, and two homologs of M. pneumoniae lipoprotein multigene family 2 were also identified immediately upstream. Homologs of mhp379 were identified in the sequenced genomes of a number of mycoplasma species, and in most cases the homologous ABC transport system was identified immediately downstream of the homologous gene; in several cases a homolog of M. pneumoniae lipoprotein multigene family 2 was also identified immediately upstream. These observations suggest that mhp379 comprises part of a conserved ABC transport operon in mycoplasmas and that the exonuclease activity of mhp379 may be associated with the conserved function of the ABC transport system in the import of nucleic acid precursors. This is the first study to identify the gene and characterize the activity of a mycoplasma exonuclease.Neither the genes nor the enzymes for the biosynthesis of nucleic acid precursors in mycoplasmas have been identified (5,21,24,34,42,46,47,51,57,59). However, genomic sequence analyses of a number of mycoplasma species have identified novel enzymes associated with salvage pathways for the biosynthesis of nucleic acids from nucleotides (5,21,24,34,42,44,51,(57)(58)(59). These salvage pathways have been functionally characterized in Mycoplasma mycoides subspecies mycoides (35-37, 58). Mycoplasma nucleases are thought to play a metabolic role in the production of nucleotide substrates from host or microbial nucleic acids released through natural and induced cell death. These nucleotide...

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Novel deoxynucleoside‐phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides

Cited by 43 publications

References 29 publications

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Purine and pyrimidine nucleotide metabolism in Mollicutes

Mycoplasma hyopneumoniae mhp379 Is a Ca ²⁺ -Dependent, Sugar-Nonspecific Exonuclease Exposed on the Cell Surface

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Novel deoxynucleoside‐phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides

Cited by 43 publications

References 29 publications

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Purine and pyrimidine nucleotide metabolism in Mollicutes

Mycoplasma hyopneumoniae mhp379 Is a Ca 2+ -Dependent, Sugar-Nonspecific Exonuclease Exposed on the Cell Surface

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Mycoplasma hyopneumoniae mhp379 Is a Ca ²⁺ -Dependent, Sugar-Nonspecific Exonuclease Exposed on the Cell Surface