Kinetics of Polynucleotide Phosphorylase: Comparison of Enzymes from Streptomyces and Escherichia coli and Effects of Nucleoside Diphosphates (original) (raw)
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ENZYMES FROM Streptomyces AND Escherichia coli AND EFFECTS OF NUCLEOSIDE 8 DIPHOSPHATES 9
Streptomyces 19 coelicolor, Streptomyces antibioticus and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3'-single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The kcat for the E. coli enzyme was ca 2-fold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3'stem-loop structure and the Km for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3'-stem-loop structure as compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 µM ADP, CDP, GDP and UDP, the Km for the phosphorolysis of the substrate with the 3'-stem-loop was some five-fold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.
Conserved domains in polynucleotide phosphorylase among eubacteria
Biochimie, 2005
Polynucleotide phosphorylase (PNPase) is a polynucleotide nucleotidyl transferase (E. C. 2.7.7.8) that is involved in mRNA degradation in prokaryotes. PNPase structure analysis has been performed in Streptomyces antibioticus; this revealed the presence of five domains: two ribonuclease PH (RPH)-like (pnp1 and pnp2), one alpha helical, one KH, and one S1 domains. The trimeric nature of this enzyme was also confirmed. In this work, we have investigated conserved domains or subdomains in bacterial PNPases (55), for this structure-based sequence homology analysis between predicted amino acid sequences from bacterial PNPases and that of S. antibioticus was performed. Our findings indicate that while pnp2 (% similarity average S = 84/% identity average I = 22), KH (S = 74.3%/I = 5.3%), S1 (S = 71.3%/I = 1.2%); and pnp1 (S = 52.8%/I = 0.3%) domain; structure and sequence are well conserved among different bacteria, alpha helical domain (S = 39.5%/I = 0) although conservation of the structure is somewhat maintained, the sequence is not conserved at all. Implications of such findings in PNPase activity will be discussed.
Quaternary Structure of Polynucleotide Phosphorylase from Escherichia coli
European Journal of Biochemistry, 1973
Casein hydrolysate and yeast extracts were purchased from Difco Laboratories, poly(A) from Miles Laboratories and Alumina 305-A from Alcoa. ADP, phenylmethylsulfonylfluoride, Tris, lactic dehydrogenase, NAD and NADH were products from Sigma, and DNAase (1 x cryst.) was from Worthington. Serum albumin, myoglobin and ovalbumin were purchased from Mann Research Laboratories and acrylamide and methylene-bis-acrylamide from Canalco. Guanidine-HC1 was from Carlo Erba (Milan, Italy) ; iodoacetic acid, maleic anhydride, and succinic anhydride were from Eastman Organic Chemicals ; yeast alcohol dehydrogenase and pyruvate kinase were from Boehringer and acrylonitrile was from Merck. Iodo[l*C]acetic acid was from The Radiochemical Centre (Amersham) and the POP, POPOP and hyamine were from Packard. E. coli aspartate transcarbamylase and RNA polymerase were respectively gifts from Drs Hervi! and Sentenac (Bio- Eur. J. Biochem. 40 (1973)
J Bacteriol, 2004
We have examined the expression of pnp encoding the 3-5-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5 ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.
The Effect of Chain Length on the Phosphorolysis of Oligonucleotides by Polynucleotide Phosphorylase
Journal of Biological Chemistry, 1970
The kinetics of phosphorolysis of compounds in the series (Ap),A (II = 2 to 8), p(Ap),A (n = 2 to 4), and (Up)I;cT, (II = 3 to 6) has been studied. K, and V,,, values were obtained for all compounds. K, decreases with increasing chain length and reaches a minimum value with compounds containing 5 phosphate residues. With the shorter compounds the dependence of K, on the nucleoside residues is also apparent. Experiments with inhibitory oligonucleotides such as (Ap),A-cyclic-p and (Ap),A-2',3'-p confirm the results with substrates. The V,,, for all oligonucleotide substrates is greater than the Vln,, for polyadenylic acid. V,,, reaches an optimum value with oligonucleotides having 5 nucleoside residues. On the basis of these results, a model for the active center of polynucleotide phosphorylase is proposed. This model has multiple subsites for the interaction of the enzyme with polynucleotides. Similar results were obtained with an enzyme form which does not require oligonucleotide primer in the polymerization reaction and an enzyme form which does require primer.
Biochimie, 2010
It has been reported that polynucleotide phosphorylase (PNPase) binds to RNA via KH and S1 domains, and at least two main complexes (I and II) have been observed in RNA-binding assays. Here we describe PNPase binding to RNA, the factors involved in this activity and the nature of the interactions observed in vitro. Our results show that RNA length and composition affect PNPase binding, and that PNPase interacts primarily with the 3 0 end of RNA, forming the complex I-RNA, which contains trimeric units of PNPase. When the 5 0 end of RNA is blocked by a hybridizing oligonucleotide, the formation of complex II-RNA is inhibited. In addition, PNPase was found to form high molecular weight (>440 kDa) aggregates in vitro in the absence of RNA, which may correspond to the hexameric form of the enzyme. We confirmed that PNPase in vitro RNA binding, degradation and polyadenylation activities depend on the integrity of KH and S1 domains. These results can explain the defective in vivo autoregulation of PNPase71, a KH point substitution mutant. As previously reported, optimal growth of a cold-sensitive strain at 18 C requires a fully active PNPase, however, we show that overexpression of a novel PNPaseDS1 partially compensated the growth impairment of this strain, while PNPase71 showed a minor compensation effect. Finally, we propose a mechanism of PNPase interactions and discuss their implications in PNPase function.
Polynucleotide Phosphorylase Activity May Be Modulated by Metabolites in Escherichia coli
Journal of Biological Chemistry, 2011
RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency.
Journal of Bacteriology, 1996
We describe the isolation and characterization of a gene (ptpA) from Streptomyces coelicolor A3(2) that codes for a protein with a deduced M r of 17,690 containing significant amino acid sequence identity with mammalian and prokaryotic small, acidic phosphotyrosine protein phosphatases (PTPases). After expression of S. coelicolor ptpA in Escherichia coli with a pT7-7-based vector system, PtpA was purified to homogeneity as a fusion protein containing five extra amino acids. The purified fusion enzyme catalyzed the removal of phosphate from p-nitrophenylphosphate (PNPP), phosphotyrosine (PY), and a commercial phosphopeptide containing a single phosphotyrosine residue but did not cleave phosphoserine or phosphothreonine. The pH optima for PNPP and PY hydrolysis by PtpA were 6.0 and 6.5, respectively. The K m values for hydrolysis of PNPP and PY by PtpA were 0.75 mM (pH 6.0, 37؇C) and 2.7 mM (pH 6.5, 37؇C), respectively. Hydrolysis of PNPP by S. coelicolor PtpA was competitively inhibited by dephostatin with a K i of 1.64 M; the known PTPase inhibitors phenylarsine oxide, sodium vanadate, and iodoacetate also inhibited enzyme activity. Apparent homologs of ptpA were detected in other streptomycetes by Southern hybridization; the biological functions of PtpA and its putative homologs in streptomycetes are not yet known.