Identification and Immunochemical Location of UMP Kinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Phosphorylation of CMP and UMP is accomplished in Bacillus subtilis, as in Escherichia coli, by two different enzymes exhibiting characteristic structural and catalytic properties. UMP kinase from B. subtilis is an oligomer whose activity is strictly dependent on GTP. The B. subtilis enzyme is unstable in the absence of UTP, which acts as an allosteric inhibitor. Antibodies raised against recombinant B. subtilis UMP kinase recognized the protein both in soluble extract and in immunoelectron microscopy. UMP kinase from B. subtilis has a peripheral distribution which is related most probably to its role in the synthesis of membrane sugar components and its putative role in cell division.     

Effects of nucleic acid compounds on viability and cell composition of Bdellovibrio bacteriovorus during starvation

The effects of various exogenous nucleic acid compounds on the viability and cell composition of Bdellovibrio bacteriovorus starved in buffer were measured. In decreasing order of effectiveness, these compounds were found to decrease the rate of loss of viability and the loss of cell carbon, cell ribonculeic acid, and cell protein: glutamate > ribonucleoside monophosphates > ribonucleosides > deoxyribonucleoside monophosphates. Similar sparing effects were not observed with nucleic acid bases, deoxyribonucleosides, ribose, ribose-5-phosphate, deoxyribose, and deoxyribose-5-phosphate. Appreciable increases in the respiration rate over the endogenous rate did not occur when cell suspensions were incubated with individual or mixtures of nucleic acid compounds. Formation of ¹⁴CO₂ by cell suspensions incubated with carbon 14-labeled nucleic acid compounds indicated ribonucleosides and ribonucleoside monophosphates were respired and to a small extent, were incorporated into cell material of non-growing cells. The respired ¹⁴CO₂ was derived mainly from the ribose portion of these molecules. No respired ¹⁴CO₂ or incorporated carbon 14 was found with bdellovibrios incubated with other nucleic acid compounds tested, including free ribose. During growth of B. bacteriovorus on Escherichia coli in the presence of exogenous UL-¹⁴C-ribonucleoside monophosphates, 10–16% of the radioactivity was in the respired CO₂ and of the radioactivity incorporated into the bdellovibrios, only 40 to 50% resided in the cell nucleic acids. However, during growth on ¹⁴C-adenine,-uracil, or-thymidine labeled E. coli, only trace amounts of ¹⁴CO₂ were found and 90% or more of the incorporated radioactivity was in the bdellovibrio nucleic acids. It is concluded that bdellovibrio can use ribonucleoside monophosphates during growth and starvation as biosynthetic precursors for synthesis of both nucleic acids and other cell materials as well as catabolizing the ribose portion for energy purposes.Abbreviations HM buffer 5 mM N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (pH 7.6) containing 0.1 mM CaCl₂ and MgCl₂ - DNA deoxyribonucleic acid - RNA ribonucleic acid - Ar, Cr, Gr, Ur ribonucleosides of adenine, cytosine, guanine, uracil, respectively - dTr deoxythymidine - AMP, CMP, GMP, UMP ribonucleoside monophosphates of adenine, cytosine, guanine, and uracil, respectively - dTMP deoxythymidine monophosphate - ATP adenosine triphosphate - PFU plaque-forming units     

Synthesis of Chiral Carbocyclic Ribonucleotides

Abstract

The carbocyclic analogs of CMP, UMP, GMP, IMP, and ribo-TMP, of the same absolute configuration as the naturally occurring β- D-ribofuranose-based ribonucleoside monophosphates, have been synthesized. The synthetic route employed Mitsunobu coupling of the heterocycles, appropriately protected where necessary, with a differentially protected, chiral carbocyclic core.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

Synthesis of chiral carbocyclic ribonucleotides.

The carbocyclic analogs of CMP, UMP, GMP, IMP, and ribo-TMP, of the same absolute configuration as the naturally occurring beta-D-ribofuranose-based ribonucleoside monophosphates, have been synthesized. The synthetic route employed Mitsunobu coupling of the heterocycles, appropriately protected where necessary, with a differentially protected, chiral carbocyclic core.     

Some properties of human erythrocyte pyrimidine 5'-nucleotidase

In haemolysates human erythrocyte pyrimidine 5'-nucleotidase had a single optimum at pH 7.2 with CMP and 6.75 with UMP as substrate. The purified enzyme showed two pH optima at pH 6.25 and 7.2 with UMP as substrate. The enzyme was inhibited by both its products - inorganic phosphate and pyrimidine nucleoside. The inhibition by inorganic phosphate appeared to be non-competitive with Ki = 1.5 mM. Contrary to previous reports adenosine and inosine did not inhibit the enzyme.     

UMP/CMPK is not the critical enzyme in the metabolism of pyrimidine ribonucleotide and activation of deoxycytidine analogs in human RKO cells

Background

Human UMP/CMP kinase was identified based on its enzymatic activity in vitro. The role of this protein is considered critical for the maintenance of pyrimidine nucleotide pool profile and for the metabolism of pyrimidine analogs in cells, based on the in vitro study of partially purified enzyme and recombinant protein. However, no detailed study has yet addressed the role of this protein in nucleotide metabolism in cells.

Methodology/Principal Findings

Two stable cell lines in which UMP/CMP kinase (mRNA: {"type":"entrez-nucleotide","attrs":{"text":"AF087865","term_id":"33150591","term_text":"AF087865"}}AF087865, EC 2.7.4.14) can be either up-regulated or down-regulated were developed using Tet-On Gene Expression Systems. The amount and enzymatic activity of UMP/CMP kinase extracted from these two cell lines can be induced up by 500% or down by 95–98%. The ribonucleotides of endogenous pyrimidine as well as the metabolism of exogenous natural pyrimidine nucleosides and their analogs were not susceptible to the altered amount of UMP/CMP kinase in these two stable RKO cell lines. The level of incorporation of pyrimidine nucleoside analogs, such as gemcitabine (dFdC) and troxacitabine (L-OddC), into cellular DNA and their potency in inhibiting cell growth were not significantly altered by up-regulation or down-regulation of UMP/CMP kinase expression in cells.

Conclusions/Significance

The UMP/CMP kinase (EC 2.7.4.14) expressed in RKO cells is not critical for the phosphorylation of (d)CMP and the maintenance of natural nucleotide pools. It also does not play an important role in the activation of dFdC and L-OddC. The increase by 500% or decrease by 95–98% in the levels of UMP/CMP kinase do not affect steady state levels of dFdC and L-OddC in RKO cells. Overall, the activity and possible mechanisms of recombinant UMP/CMP kinase expressed in the in vitro system can not be extended to that of UMP/CMP kinase expressed in a cell system or an in vivo system.     

Pyrimidine ribonucleoside catabolism in Pseudomonas fluorescens biotype A

The pyrimidine ribonucleosides uridine or cytidine were shown to serve as a source of nitrogen or carbon for the growth of Pseudomonas fluorescens strain A126. After incubation of either pyrimidine ribonucleoside with extracts of this strain, the resultant catabolic products were detected by thin-layer chromatography. It was found that pyrimidine ribonucleoside catabolism in this pseudomonad involved the enzymes nucleoside hydrolase and cytosine deaminase. The specific activities of both these enzymes could be influenced by the nitrogen or carbon source present in the medium.     

Sequence analysis of the Brevibacterium lactofermentum trp operon

Summary Brevibacterium lactofermentum, a Gram-positive bacterium, is a commercially important amino acid producer. In this organism, the tryptophan biosynthetic enzymes are encoded within a 7725 bp HapII-BamHI fragment. Seven open reading frames were identified as trp genes by complementation tests with various B. lactofermentum and Escherichia coli tryptophan auxotrophs. Following the nomenclature established for E. coli and Serratia marcescens, the B. lactofermentum trp genes were designated trpL, trpE, trpG, trpD, trpC (including the trpF domain), trpB, and trpA. The organization of these genes is identical to that in S. marcescens. The nucleotide sequences of the putative ribosome-binding sites for the B. lactofermentum trp genes resemble those of E. coli and Bacillus subtilis. Computer analysis revealed that the trp enzymes of B. lactofermentum resemble the enzymes of the Gram-negative E. coli more closely than those of the Gram-positive B. subtilis.Abbreviations bp base pairs - kb kilobases     

Kinetics and equilibria of pyrimidine nucleoside monophosphate kinase from human erythrocytes.

The common type of pyrimidine nucleoside monophosphate kinase (ATP:CMP phosphotransferase, EC 2.7.4.14), purified 50 000-fold from human erythrotes, reacted with a wide variety of nucleotides, but only ATP, dATP, UMP and CMP were good substrates. The optimum Mg2+ concentration, 2-3 mM, was generally independent of substrate concentration, of the nature of the substrate, and of the direction of the reaction. Kinetic studies indicated that a ternary complex was formed, that the substrates were bound at two unlike sites, and that the order of addition of substrates was random. Equilibrium constants were ATP + UMP 0.98, ATP + CMP 1.59, dATP + UMP 1.13, and ATP + AMP 1.20.     

Enzymatic Characterization of AMP Phosphorylase and Ribose-1,5-Bisphosphate Isomerase Functioning in an Archaeal AMP Metabolic Pathway

AMP phosphorylase (AMPpase), ribose-1,5-bisphosphate (R15P) isomerase, and type III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to constitute a novel pathway involved in AMP metabolism in the Archaea. Here we performed a biochemical examination of AMPpase and R15P isomerase from Thermococcus kodakarensis. R15P isomerase was specific for the α-anomer of R15P and did not recognize other sugar compounds. We observed that activity was extremely low with the substrate R15P alone but was dramatically activated in the presence of AMP. Using AMP-activated R15P isomerase, we reevaluated the substrate specificity of AMPpase. AMPpase exhibited phosphorylase activity toward CMP and UMP in addition to AMP. The [S]-v plot (plot of velocity versus substrate concentration) of the enzyme toward AMP was sigmoidal, with an increase in activity observed at concentrations higher than approximately 3 mM. The behavior of the two enzymes toward AMP indicates that the pathway is intrinsically designed to prevent excess degradation of intracellular AMP. We further examined the formation of 3-phosphoglycerate from AMP, CMP, and UMP in T. kodakarensis cell extracts. 3-Phosphoglycerate generation was observed from AMP alone, and from CMP or UMP in the presence of dAMP, which also activates R15P isomerase. 3-Phosphoglycerate was not formed when 2-carboxyarabinitol 1,5-bisphosphate, a Rubisco inhibitor, was added. The results strongly suggest that these enzymes are actually involved in the conversion of nucleoside monophosphates to 3-phosphoglycerate in T. kodakarensis.     

Study of the Escherichia coli tRNA nucleotidyltransferase. Specificity of the enzyme for nucleoside triphosphates

Besides the main reactions leading to the repair of tRNA molecules deprived of part or all of their 3′ terminal -pCpCpA sequence, purified E. coli tRNA nucleotidyltransferase catalyzes in vitro, under certain conditions the synthesis of sequences not found in natural tRNAs. In the absence of CTP, AMP is incorporated directly into tRNA-pX or tRNA-pXpC leading to tRNA-pXpA or tRNA-pXpCpA respectively. In the absence of ATP one extra CMP is added to tRNA-pXpCpC to form tRNA-pXpCpCpC. UMP can be incorporated instead of CMP and the sequence -pXpU and -pXpCpU formed. The incorporation of UMP cannot be followed by the incorporation of either a second UMP or an AMP. In all cases, the rate of misincorporation is lower than the rate of the synthesis of the normal sequence.The apparent K_M of the enzyme for UTP is 3.0 10⁻⁴ M. CTP inhibits competitively the incorporation of UMP into tRNA-pX with a K_i value (1.6 10⁻⁵ M) close to its apparent K_M.     

Nucleotides modulate the activity of aspartate racemase of Scapharca broughtonii

The activity of -aspartate racemase purified from Scapharca broughtonii has been found to depend markedly on some nucleotides. Purine nucleoside monophosphates enhanced the enzyme activity, which was, on the contrary, lowered by purine nucleoside triphosphates and not affected by pyrimidine nucleotides. AMP produced the highest increase of seven-fold in the enzyme activity at 6 mM and a half-maximum increase at approximately 3.8 mM. ATP caused a half-maximum decrease in the activity at approximately 1.4 mM and the remaining activity was lower than 7% at saturating ATP concentrations. AMP and ATP both brought about changes in V_max and not in K_m. Analysis of the effect of AMP and ATP suggests that each of them has its own primary binding site, which is different from the substrate-binding site. In view of these effects of the nucleotides, the roles of the racemase and -aspartate in energy metabolism under anoxic conditions are discussed.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by nonionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.Abbreviations ISP intracellular serine protease - ISP-A-Bsu A-50 and ISP-B-Bsu A-50 molecular forms A and B of B. subtilis A-50 intracellular serine protease, respectively - SDS sodium dodecyl sulfate - PMSF phenylmethyl sulfonylfluoride - pNA p-nitroanilide - Buffer A 50 mM Tris-(hydroxymethyl)aminomethane-1 mM CaCl₂ adjusted to pH 8.5 with HCl     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Rabbit liver dCMP phosphohydrolase: a pyrimidine 5'-nucleotidase I-like enzyme in non-erythrocytic cells

A nucleotide phosphomonoesterase activity that preferably hydrolyzed dCMP was detected in rabbit liver and purified approximately 20-fold. The enzyme was similar in the catalytic and molecular properties to pyrimidine 5'-nucleotidase subclass I (P5N-I), which distributed specifically in vertebrate erythrocytes. In addition to liver, the activity was found in rabbit kidney, spleen, heart, intestine, but was not detected in any rat or chicken tissues tested. The rabbit enzyme protein reacted with antibodies against chicken P5N-I. Its pI was estimated to be approximately 5.3, and the enzyme was concluded to consist of single polypeptide of an approximately 38 kDa based on gel filtration and Western blot analysis. The partially purified enzyme preferentially hydrolyzes dCMP, UMP and CMP, K(m) values for these substrates are approximately 0.3 mM, the optimal pH is approximately 7, and the enzyme requires Mg(2+). This nucleotidase may contribute to the regulation of intracellular pyrimidine nucleotides in the rabbit.     

Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.