Bacterial DNA ligases

DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell. All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor. All Bacteria (eubacteria) contain NAD+-dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics. In addition to their NAD+-dependent enzymes, some Bacteria contain genes for putative ATP-dependent DNA ligases. The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism. The putative ATP-dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses. Phylogenetic analysis suggests that all NAD+-dependent DNA ligases are closely related, but the ATP-dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.     

Changes in germ cell adenylate cyclase and protein carboxyl methylase activities in rat testicular tissue during bilateral cryptorchidism and after orchidopexy

Soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase are two enzymes that are primarily localized in haploid germ cels of rat testicular tissue, and both enzymes exhibit an increase in activity in association with sexual maturation. Experimental cryptorchidism (surgery at 17 days of age) in immature rats prevented the age-dependent increase in the activity of these two testicular enzymes. After orchidopexy at 34 days of age the activities of these two enzymes increased to normal control values in association with testicular growth. These observations show that biochemical markers such as soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase can be used to follow germ cell differentiation.     

Occurrence of 3-hydroxyretinol in the freshwater fish Bagarius bagarius and Wallago attu. Isolation and synthesis.

1. Four different types of alpha-mannosidase activity were shown to occur in several tissues from the rat. There is the Zn2+-dependent enzyme, active at acidic pH, and three enzymes that are active near to neutral pH. 2. The 'neutral' enzymes are activated by Fe2+, Co2+ or Mn2+. 3. Optimum activities for these three enzymes are shown at pH values of 5.2, 6.5 and 7.3. The activity at pH6.5 is the only one evident without metal-ion activation, but activity is enhanced by all three metal ions. The activity at pH 5.2 is seen only in the presence of Fe2+ or Co2+, and the activity at pH7.3 is seen only in the presence of Co2+ or Mn2+ and in a non-chelating buffer medium. 4. The pH6.5-active enzyme is inactivated by EDTA, but activity is restored by excess of metal ion. 5. The enzymes differ markedly in their stability. The pH6.5-active enzyme is very labile and the pH7.3-active enzyme is the most stable. 6. Tissue preparations vary widely in their activity at pH6.5, but where activity is low it can be increased by incubation with one of the activating metal cations. 7. All the enzymes active at neutral pH are inhibited by heavy-metal ions and stabilized to some extent by thiol groups.     

Two ribonucleases H from cultured plant cells.

Two forms of enzyme with ribonuclease H (RNase H) [EC 3.1.4.34] activities, have been partially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+-dependent RNase H, and the second is an Mg2+-dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2 M and 0.4 M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+-dependent RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15 mM Mg2+ for maximal activity, whereas the Mn2+-dependent enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4 mM, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. The apparent molecular weight of the Mg2+-dependent RNase H was estimated to 18--20 X 10(4) and that of the Mn 2+- dependent RNase H was estimated to be 14 x 10(4) by gel filtration.     

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.     

Distinct enzymic functional groups are required for the phosphomonoesterase and phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase/phosphatase

The central phosphatase domain of Clostridium thermocellum polynucleotide kinase/phosphatase (CthPnkp) belongs to the dinuclear metallophosphoesterase superfamily. Prior mutational studies of CthPnkp identified 7 individual active site side chains (Asp-187, His-189, Asp-233, Asn-263, His-323, His-376, and Asp-392) required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Here we find that Mn2+-dependent phosphomonoesterase activity requires two additional residues, Arg-237 and His-264. We report that CthPnkp also converts bis-p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate via a processive two-step mechanism. The Ni2+-dependent phosphodiesterase activity of CthPnkp requires the same seven side chains as the Ni2+-dependent phosphomonoesterase. However, the Mn2+-dependent phosphodiesterase activity does not require His-189, Arg-237, or His-264, each of which is critical for the Mn2+-dependent phosphomonoesterase. Mutations H189A, H189D, and D392N transform the metal and substrate specificity of CthPnkp such that it becomes a Mn2+-dependent phosphodiesterase. The H189E change results in a Mn2+/Ni2+-dependent phosphodiesterase. Mutations H376N, H376D, and D392E convert the enzyme into a Mn2+-dependent phosphodiesterase-monoesterase. The phosphodiesterase activity is strongly stimulated compared with wild-type CthPnkp when His-189 is changed to Asp, Arg-237 is replaced by Ala or Gln, and His-264 is replaced by Ala, Asn, or Gln. Steady-state kinetic analysis of wild-type and mutated enzymes illuminates the structural features that affect substrate affinity and kcat. Our results highlight CthPnkp as an "undifferentiated" diesterase-monoesterase that can evolve toward narrower metal and substrate specificities via alterations of the active site milieu.     

Two malic enzymes in Pseudomonas aeruginosa

Cell-free extract supernatant fluids of Pseudomonas aeruginosa were shown to lack malic dehydrogenase but possess a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent enzymatic activity, with properties suggesting a malic enzyme (malate + NAD (NADP) --> pyruvate + reduced NAD (NADH) (reduced NADP [NADPH] + CO(2)), in agreement with earlier findings. This was confirmed by determining the nature and stoichiometry of the reaction products. Differences in heat stability and partial purification of these activities demonstrated the existence of two malic enzymes, one specific for NAD and the other for NADP. Both enzymes require bivalent metal cations for activity, Mn(2+) being more effective than Mg(2+). The NADP-dependent enzyme is activated by K(+) and low concentrations of NH(4) (+). Both reactions are reversible, as shown by incubation with pyruvate, CO(2), NADH, or NADPH and Mn(2+). The molecular weights of the enzymes were estimated by gel filtration (270,000 for the NAD enzyme and 68,000 for the NADP enzyme) and by sucrose density gradient centrifugation (about 200,000 and 90,000, respectively).     

Effects of variation in the structure of spermine on the association with DNA and the induction of DNA conformational changes.

Manganese shares the uniport mechanism of mitochondrial calcium influx, accumulates in mitochondria and is cleared only very slowly from brain. Using dual-label isotope techniques, we have investigated both Mn2+ and Ca2+ mitochondrial efflux kinetics. We report that (1) there is no significant Na(+)-dependent Mn2+ efflux from brain mitochondria; (2) Mn2+ inhibits both Na(+)-dependent and Na(+)-independent Ca2+ efflux in brain, in a mode that appears to be primarily competitive and with apparent Ki values of 5.1 and 7.9 nmol/mg respectively; and (3) Ca2+ does not appear to inhibit Mn2+ efflux from brain mitochondria. Findings (1) and (2) suggest the possibility of mitochondrial accumulation of both Mn2+ and Ca2+ in Mn2(+)-intoxicated brain.     

Identification and characterization of Mg2+-dependent phosphotyrosyl protein phosphatase from rat liver cytosol

Although highly purified preparations of Mg2+-dependent phosphoseryl protein phosphatase (also designated phosphatase IA or phosphatase 2C) dephosphorylated phosphotyrosyl histone, the activity has been resolved from phosphatase IA by polyacrylamide gel electrophoresis at pH 9.5. This novel phosphotyrosyl-specific protein phosphatase absolutely requires Mg2+ or Mn2+ for activity, is inhibited by Zn2+, vanadate and fluoride, and has an optimal pH of 9.0 and Mr = 50,000. Certain properties of this phosphatase so closely resemble those of phosphatase IA that the two enzymes tend to be copurified through various separation procedures.     

Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification

Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.     

Independent bindings of Mn2+ and Mg2+ to the active site of B. cereus glutamine synthetase

Glutamine synthetase purified from Bacillus cereus IFO 3131 was modified by iodoacetamide and the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Only Mg2+-dependent activity was inactivated by iodoacetamide, whereas only Mn2+-dependent activity was inactivated by FSBA. When iodoacetamide-treated enzyme was reacted with FSBA, Mn2+-dependent activity was also inactivated. Mg2+ plus Mn2+-dependent activity was inactivated in any case. The results suggested that the binding sites of Mn2+ and Mg2+ are separate from each other in the active site of B. cereus glutamine synthetase and that bindings of Mg2+ and Mn2+ to each site are required for normal activity in vivo.     

The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen

NRAMPs (natural resistance-associated macrophage proteins) have been characterized in mammals as divalent transition metal transporters involved in iron metabolism and host resistance to certain pathogens. The mechanism of pathogen resistance is proposed to involve sequestration of Fe2+ and Mn2+, cofactors of both prokaryotic and eukaryotic catalases and superoxide dismutases, not only to protect the macrophage against its own generation of reactive oxygen species, but to deny the cations to the pathogen for synthesis of its protective enzymes. NRAMP homologues are also present in bacteria. We report the cloning and characterization of the single NRAMP genes in Escherichia coli and Salmonella enterica ssp. typhimurium, and the cloning of two distinct NRAMP genes from Pseudomonas aeruginosa and an internal fragment of an NRAMP gene in Burkholderia cepacia. The genes are designated mntH because the two enterobacterial NRAMPs encode H+-stimulated, highly selective manganese(II) transport systems, accounting for all Mn2+ uptake in each species under the conditions tested. For S. typhimurium MntH, the Km for 54Mn2+ ( approximately 0.1 microM) was pH independent, but maximal uptake increased as pH decreased. Monovalent cations, osmotic strength, Mg2+ and Ca2+ did not inhibit 54Mn2+ uptake. Ni2+, Cu2+ and Zn2+ inhibited uptake with Kis greater than 100 microM, Co2+ with a Ki of 20 microM and Fe2+ with a Ki that decreased from 100 microM at pH 7. 6 to 10 microM at pH 5.5. Fe3+ and Pb2+ inhibited weakly, exhibiting Kis of 50 microM, while Cd2+ was a potent inhibitor with a Ki of about 1 microM. E. coli MntH had a similar inhibition profile, except that Kis were three- to 10-fold higher. Both S. typhimurium and E. coli MntH also transport 55Fe2+ however, the Kms are equivalent to the Kis for Fe2+ inhibition of Mn2+ uptake, and are thus too high to be physiologically relevant. In both S. typhimurium and E. coli, mntH:lacZ constructs were strongly induced by hydrogen peroxide, weakly induced by EDTA and unresponsive to paraquat, consistent with the presence of Fur and OxyR binding sites in the promoters. Strains overexpressing mntH were more susceptible to growth inhibition by Mn2+ and Cd2+ than wild type, and strains lacking a functional mntH gene were more susceptible to killing by hydrogen peroxide. In S. typhimurium strain SL1344, mntH mutants showed no defect in invasion of or survival in cultured HeLa or RAW264.7 macrophage cells; however, expression of mntH:lacZ was induced severalfold by 3 h after invasion of the macrophages. S. typhimurium mntH mutants showed only a slight attenuation of virulence in BALB/c mice. Thus, the NRAMP Mn2+ transporter MntH and Mn2+ play a role in bacterial response to reactive oxygen species and possibly have a role in pathogenesis.     

Identification and biochemical characterization of a eukaryotic-type serine/threonine kinase and its cognate phosphatase in Streptococcus pyogenes: their biological functions and substrate identification

A eukaryotic-type signaling system in group A Streptococcus (GAS) was identified and characterized. This system comprises primarily the products of two co-transcribed genes, a eukaryotic-type Ser/Thr kinase (SP-STK) and phosphatase (SP-STP) and their endogenous substrate histone-like protein (SP-HLP). Enzyme activities of SP-STK and SP-STP primarily depended on Mn(2+). The site on the substrate for reversible phosphorylation by these enzymes was found to be only the threonine residue. Using specific antibodies generated against these proteins, SP-STK was found to be membrane-associated with its N-terminal kinase domain facing the cytoplasm and its C-terminal repeat domain outside the membrane and cell-wall associated. Further, SP-STP, primarily a cytoplasmic protein, was found to be a major secretory protein of GAS and essential for bacterial survival. Three isogenic mutants, lacking either the entire SP-STK, or one of its two domains, were found displaying distinct pleiotropic effects on growth, colony morphology, cell division/septation, surface protein/virulence factor expression, bacterial ability to adhere to and invade human pharyngeal cells, and resist phagocytosis by human neutrophils. In addition to these properties, the ability of these three proteins to modulate the expression of the major virulence factors, the M protein and the capsule, indicates that these proteins are structurally and functionally distinct from the kinases and phosphatases described in other microorganisms and play a key role in GAS pathogenesis.     

Manganese uptake and transportation as well as antioxidant response to excess manganese in plants]

Manganese (Mn) is an essential micronutrient throughout all stages of plant development. Mn plays an important role in many metabolic processes in plants. It is of particular importance to photosynthetic organisms in the chloroplast of which a cluster of Mn atoms at the catalytic centre function in the light-induced water oxidation by photosystem II, and also function as a cofactor for a variety of enzymes, such as Mn-SOD. But excessive Mn is toxic to plants which is one of the most toxic metals in acid soils. The knowledge of Mn(2+) uptake and transport mechanisms, especially the genes responsible for transition metal transport, could facilitate the understanding of both Mn tolerance and toxicity in plants. Recently, several plant genes were identified to encode transporters with Mn(2+) transport activity, such as zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, natural resistance-associated macrophage protein (Nramp) transporters, cation/H(+) antiporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPase. In addition, excessive Mn frequently induces oxidative stress, then several defense enzymes and antioxidants are stimulated to scavenge the superoxide and hydrogen peroxide formed under stress. Mn-induced oxidative stress and anti-oxidative reaction are very important mechanisms of Mn toxicity and Mn tolerance respectively in plants. This article reviewed the transporters identified as or proposed to be functioning in Mn(2+) transport, Mn toxicity-induced oxidative stress, and the response of antioxidants and antioxidant enzymes in plants to excessive Mn to facilitate further study. Meanwhile, basing on our research results, new problems and views are brought forward.     

Metal-ion-dependent catalysis and specificity of CCA-adding enzymes: a comparison of two classes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases] catalyze synthesis of the conserved and essential CCA sequence to the tRNA 3' end. These enzymes are divided into two classes of distinct structures that differ in the overall orientation of the head to tail domains. However, the catalytic core of the two classes is conserved and contains three carboxylates in a geometry commonly found in DNA and RNA polymerases that use the two-metal-ion mechanism for phosphoryl transfer. Two important aspects of the two-metal-ion mechanism are tested here for CCA enzymes: the dependence on metal ions for catalysis and for specificity of nucleotide addition. Using the archaeal Sulfolobus shibabae enzyme as an example of the class I, and the bacterial Escherichia coli enzyme as an example of the class II, we show that both enzymes depend on metal ions for catalysis, and that both use primarily Mg2+ and Mn2+ as the "productive" metal ions, but several other metal ions such as Ca2+ as the "nonproductive" metal ions. Of the two productive metal ions, Mg2+ specifically promotes synthesis of the correct CCA, whereas Mn2+ preferentially accelerates synthesis of the noncognate CCC and poly(C). Thus, despite evolution of structural diversity of two classes, both classes use metal ions to determine catalysis and specificity. These results provide critical insights into the catalytic mechanism of CCA synthesis to allow the two classes to be related to each other, and to members of the larger family of DNA and RNA polymerases.     

The in vitro uptake of glutamate in GLAST and GLT-1 transfected mutant CHO-K1 cells is inhibited by manganese

In the central nervous system (CNS), extracellular concentrations of amino acids (e.g., aspartate, glutamate) and divalent metals (e.g., zinc, copper, manganese) are primarily regulated by astrocytes. Adequate glutamate homeostasis and control over extracellular concentrations of these excitotoxic amino acids are essential for the normal functioning of the brain. Not only is glutamate of central importance for nitrogen metabolism but, along with aspartate, it is the primary mediator of excitatory pathways in the brain. Similarly, the maintenance of proper Mn levels is important for normal brain function. Brain glutamate is removed from the extracellular fluid mainly by astrocytes via high affinity astroglial Na+-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of Mn on specific glutamate transporters have yet to be determined. As a first step in this process, we examined the effects of Mn on the transport of [D-2, 3-3H]D-aspartate, a non-metabolizable glutamate analog, in Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) or GLT-1 (EAAT2). Mn-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was pronounced in both the GLT-1 and GLAST transfected cells. This resulted in a statistically significant inhibition (p<0.05) of glutamate uptake compared with transfected control in the absence of Mn treatment. These studies suggest that Mn accumulation in the CNS might contribute to dysregulation of glutamate homeostasis.     

Paradoxical effects of copper and manganese on brain mitochondrial function

Defects in the mitochondrial genome have been associated with Parkinson's and Alzheimer's disease, and apoptosis can be triggered by the presence of energetically compromised mitochondria. Thus, in this study we have examined whether the divalent cations Cu2+ and Mn2+ could influence mitochondrial function in vitro. Mitochondrial electron transport was dose and time dependently reduced by Cu2+ to a greater extent with succinate as a substrate. Following a 60 min preincubation period, Mn2+ dose dependently inhibited electron transport to a greater extent with lactate and malate. In contrast, paradoxical effects were seen following a 5 min preincubation period with Mn2+. Cu2+ dose-dependently reduced NADH-dependent lactate dehydrogenase (LDH) activity, with almost complete inhibition apparent at 10 microM. An initial induction of LDH by 10 microM Mn2+ was partially reversed by higher concentrations of the metal. Cu2+ dose-dependently reduced flavin adenine dinucleotide (FAD)-dependent monoamine oxidase A (MAO-A) activity in a time-independent manner, with an IC50 value approximately 20 microM, whereas Mn2+ had no effect. In conclusion, it is proposed that Cu2+ and Mn2+ have differential effects on nicotinamide adenine dinucleotide (NAD) and FAD-dependent mitochondrial enzymes at the level of the essential cofactors. Cu2+ appears to exert an inhibitory effect on both NAD and FAD-dependent enzymes, but predominantly against the latter, including MAO-A and succinate dehydrogenase. The complex responses to Mn2+ may be due to dose-related effects on the interconversion of NAD and NADH and reversible enzymatic reactions employing this nucleotide cofactor.     

Differential effects of metal ions on Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase and stoichiometric incorporation of HCO3- into a cobalt(III)--enzyme complex.

Mg2+ or Mn2+ ions supported both the carboxylase and oxygenase activities of the Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase. For the carboxylase reaction, Mn2+ supported 25% of the maximum activity obtained with Mg2+; oxygenase activity, however, was twice as great with Mn2+ as compared to that with Mg2+. A further differential effect was obtained with Co2+. Co2+ did not support carboxylase activity and, in fact, was a strong inhibitor of Mg2+-dependent carboxylase activity, with a Ki of 10 microM. Co2+ did, however, support oxygenase activity, eliciting about 40% of the Mg2+-dependent oxygenase activity. No other divalent cations supported either activity. With high concentrations of Mg2+ or Mn2+, maximum carboxylase activity was seen after a 5-min activation period; activity decreased to about half of maximum after 30-min activation. A similar time dependence of activation was observed with Mn2+-dependent oxygenase activity but was not seen for Mg2+- or Co2+-dependent activity. Both carboxylase and oxygenase activities were inactivated by the oxidation of Co2+ to Co(III) with the resultant formation of a stable Co(III)--enzyme complex. In the presence of HCO3- (CO2), Co(III) modification was stoichiometric, with two cobalt atoms bound per enzyme dimer. Carbon dioxide was also incorporated into this Co(III)--enzyme complex, but only one molecule per enzyme dimer was bound, indicative of half-the-sites activity. These results thus indicate that there are substantial differences in the metal ion sites of the carboxylase and oxygenase activities of R, rubrum ribulosebisphosphate carboxylase/oxygenase.     

Purification and characterization of the Ca2+ plus Mg2+-dependent endodeoxyribonuclease from calf thymus chromatin

Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.