Purification of a non-specific nucleoside hydrolase from Alaska pea seeds.

A non-specific nucleoside hydrolase has been isolated from germinated Alaska pea seeds. The enzyme catalyzes the hydrolysis of both purines and pyrimidines along with ribo- and deoxyribonucleosides. A purification scheme utilized ammonium sulfate precipitation, ion exchange chromatography and size exclusion chromatography, resulted in 103-fold purification with a recovery of 2.8%. The purified protein has a specific activity of 0.308 µmol/min•mg. The subunit molecular weight was 26103 Da and the enzyme exists as a dimer. The enzyme retains a significant amount of activity over a wide pH range with the maximum activity occurring at a pH of 6.0. The maximum activity was observed with adenosine as the substrate followed by inosine and guanosine, respectively. The Km for adenosine was 184 ±â€¯34 µM and for inosine 283 ±â€¯88 µM. In addition to the nucleoside hydrolase activity, adenosine deaminase activity was seen in the initial extract. Using adenosine as the substrate with the initial extract from the germinated seeds, the products adenine, inosine, and hypoxanthine were identified based on their retention times during reverse phase HPLC.

Expression, purification and spectrophotometric analysis of nucleoside hydrolase from Leishmania chagasi (LcNH).

Leishmaniasis represents an important public health problem in several countries. The main target in this study is the nucleoside hydrolase Leishmania chagasi (LcNH) that is responsible for causing visceral leishmaniasis, principally in Brazil. Nucleoside hydrolase enzymes are members of this pathway, hydrolyzing the N-glycosidic bond of ribonucleosides for the synthesis of nucleic acids. We present here for the first time, the expression and purification protocols to obtain the enzymes LcNH1 and LcNH2 that can be employed to explore novel strategies to produce nucleoside hydrolase inhibitors for use in chemotherapy. Protein integrity was also confirmed by SDS-PAGE gel, mass spectrometry and enzymatic activity.

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay.

S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) are an essential superfamily of enzymes that catalyze the transfer of a methyl group to several biomolecules. Alterations in the methylation of cellular components crucially impact vital biological processes, making MTases attractive drug targets for treating infectious diseases and diseases caused by overactive human-encoded MTases. Several methods have been developed for monitoring the activity of MTases, but most MTase assays have inherent limitations or are not amenable for high-throughput screening. We describe a universal, competitive fluorescence polarization (FP) assay that directly measures the production of S-adenosylhomocysteine (AdoHcy) from MTases. Our developed assay monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy molecule complexed to a catalytically inert 5'-methylthioadenosine nucleosidase (MTAN-D198N) variant performed in a mix-and-read format. Producing the fluorescently labeled molecule involves a one-pot synthesis by combining AdoHcy with an amine-reactive rhodamine derivative, which possesses a Kd value of 11.3 ± 0.7 nM to MTAN-D198N. The developed competitive FP assay expresses a limit of detection for AdoHcy of 6 nM and exhibits a 34-fold preference to AdoHcy in comparison to AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen with the NIH Clinical Collection as well as determining the kinetic parameters of l-histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the developed assay is applicable to other AdoMet-dependent and ATP-dependent enzymes by detecting various adenosine-containing molecules including 5'-methylthioadenosine, AMP, and ADP.

Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 µM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate.

Crystal Structures of Mycobacterium tuberculosis CysQ, with Substrate and Products Bound.

In many organisms, 3'-phosphoadenosine 5'-phosphate (PAP) is a product of two reactions in the sulfur activation pathway. The sulfurylation of biomolecules, catalyzed by sulfotransferases, uses 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor, producing the sulfated biomolecule and PAP product. Additionally, the first step in sulfate reduction for many bacteria and fungi reduces the sulfate moiety of PAPS, producing PAP and sulfite, which is subsequently reduced to sulfide. PAP is removed by the phosphatase activity of CysQ, a 3',5'-bisphosphate nucleotidase, yielding AMP and phosphate. Because excess PAP alters the equilibrium of the sulfur pathway and inhibits sulfotransferases, PAP concentrations can affect the levels of sulfur-containing metabolites. Therefore, CysQ, a divalent cation metal-dependent phosphatase, is a major regulator of this pathway. CysQ (Rv2131c) from Mycobacterium tuberculosis (Mtb) was successfully expressed, purified, and crystallized in a variety of ligand-bound states. Here we report six crystal structures of Mtb CysQ, including a ligand-free structure, a lithium-inhibited state with substrate PAP bound, and a product-bound complex with AMP, phosphate, and three Mg(2+) ions bound. Comparison of these structures together with homologues of the superfamily has provided insight into substrate specificity, metal coordination, and catalytic mechanism.

Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli.

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480µM. No activity was exhibited against 2'-OH and 3'-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5'-OH group or with the 2'-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.

Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv.

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease's causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli's nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (Ea, ΔG(#), ΔS(#), ΔH(#)) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.

Expression and purification of active mouse and human NEIL3 proteins.

Endonuclease VIII-like 3 (Neil3) is one of the five DNA glycosylases found in mammals that recognize and remove oxidized bases, and initiate the base excision repair (BER) pathway. Previous attempts to express and purify the mouse and human orthologs of Neil3 in their active form have not been successful. Here we report the construction of bicistronic expression vectors for expressing in Escherichia coli the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3Δ324), as well as the glycosylase domain of human Neil3 (NEIL3Δ324). The purified Neil3 proteins are all active, and NEIL3Δ324 exhibits similar glycosylase/lyase activity as MmuNeil3Δ324 on both single-stranded and double-stranded substrates containing thymine glycol (Tg), spiroiminodihydantoin (Sp) or an abasic site (AP). We show that N-terminal initiator methionine processing is critical for the activity of both mouse and human Neil3 proteins. Co-expressing an E. coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNeil3, MmuNeil3Δ324 and NEIL3Δ324 improves the N-terminal methionine processing and increases the percentage of active Neil3 proteins in the preparation. The purified Neil3 proteins are suitable for biochemical, structural and functional studies.

Microfluidic glycosyl hydrolase screening for biomass-to-biofuel conversion.

The hydrolysis of biomass to fermentable sugars using glycosyl hydrolases such as cellulases and hemicellulases is a limiting and costly step in the conversion of biomass to biofuels. Enhancement in hydrolysis efficiency is necessary and requires improvement in both enzymes and processing strategies. Advances in both areas in turn strongly depend on the progress in developing high-throughput assays to rapidly and quantitatively screen a large number of enzymes and processing conditions. For example, the characterization of various cellodextrins and xylooligomers produced during the time course of saccharification is important in the design of suitable reactors, enzyme cocktail compositions, and biomass pretreatment schemes. We have developed a microfluidic-chip-based assay for rapid and precise characterization of glycans and xylans resulting from biomass hydrolysis. The technique enables multiplexed separation of soluble cellodextrins and xylose oligomers in around 1 min (10-fold faster than HPLC). The microfluidic device was used to elucidate the mode of action of Tm_Cel5A, a novel cellulase from hyperthermophile Thermotoga maritima . The results demonstrate that the cellulase is active at 80 °C and effectively hydrolyzes cellodextrins and ionic-liquid-pretreated switchgrass and Avicel to glucose, cellobiose, and cellotriose. The proposed microscale approach is ideal for quantitative large-scale screening of enzyme libraries for biomass hydrolysis, for development of energy feedstocks, and for polysaccharide sequencing.

Purification, characterization, and functional properties of a novel glycoprotein from tartary buckwheat (Fagopyrum tartaricum) seed.

A pure glycoprotein (BGP4-I) was obtained from tartary buckwheat seeds by aqueous extraction followed by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The average molecular weight of BGP4-I, as determined by high performance gel permeation chromatography, was 123.43 kDa. The structure of BGP4-I was characterized based on Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy, etc. Based on the nano-liquid chromatography-coupled electrospray ionization mass spectrometry analysis of the amino acid sequence of BGP4-I, belongs unequivocally to the glycosyl hydrolase family 1 in the Carbohydrate Active Enzymes database by alignment studies. The specific activity of BGP4-I was 18.44 µmol/min/mg on the substrate p-nitrophenyl-ß-d-glucopyranoside. Furthermore, BGP4-I is unique in its specificity for some substrates. These results suggest that the BGP4-I from tartary buckwheat seeds is a novel specific ß-glucosidase setting the foundation for potential applications in the food industry.

Expression and purification of NEIL3, a human DNA glycosylase homolog.

The base excision repair (BER) pathway is mainly responsible for the repair of a vast number of non-bulky lesions produced by alkylation, oxidation or deamination of bases. DNA glycosylases are the key enzymes that recognize damaged bases and initiate BER by catalyzing the cleavage of the N-glycosylic bond between the base and the sugar. Many of the mammalian DNA glycosylases have been identified by a combination of biochemical and bioinformatics analysis. Thus, a mammalian family of three proteins (NEIL1, NEIL2 and NEIL3) that showed homology to the Escherichia coli Fpg/Nei DNA glycosylases was identified. Two of the proteins, NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of a diverse number of oxidized lesions. However, much less is known about NEIL3. The biochemical properties of NEIL3 have not been elucidated. This is mainly due to the difficulty in the expression and purification of NEIL3. Here, we describe the expression and partial purification of full-length human NEIL3 and the expression, purification and characterization of a truncated human core-NEIL3 (amino acids 1-301) that contains the complete E. coli Fpg/Nei-like domain but lacks the C-terminal region.

Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1.

Human ADP-ribosylhydrolase 1 (hARH1, ADPRH) cleaves the glycosidic bond of ADP-ribose attached to an Arg residue of a protein. hARH1 has been cloned, expressed heterologously in Escherichia coli, purified and crystallized in complex with K(+) and ADP. The orthorhombic crystals contained one monomer per asymmetric unit, exhibited a solvent content of 43% and diffracted X-rays to a resolution of 1.9 A. A prerequisite for obtaining well diffracting crystals was the performance of X-ray fluorescence analysis on poorly diffracting apo hARH1 crystals, which revealed the presence of trace amounts of K(+) in the crystal. Adding K-ADP to the crystallization cocktail then resulted in a crystal of different morphology and with dramatically improved diffraction properties.

Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus--biochemical characterization and homology modeling.

We report the characterization of the pyrimidine-specific ribonucleoside hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsCU-NH). The gene SSO0505 encoding SsCU-NH was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. SsCU-NH is a homotetramer of 140 kDa that recognizes uridine and cytidine as substrates. SsCU-NH shares 34% sequence identity with pyrimidine-specific nucleoside hydrolase from E. coli YeiK. The alignment of the amino acid sequences of SsCU-NH with nucleoside hydrolases whose 3D structures have been solved indicates that the amino acid residues involved in the calcium- and ribose-binding sites are preserved. SsCU-NH is highly thermophilic with an optimum temperature of 100 degrees C and is characterized by extreme thermodynamic stability (T(m) = 106 degrees C) and kinetic stability (100% residual activity after 1 h incubation at 90 degrees C). Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. The structure of the enzyme determined by homology modeling provides insight into the proteolytic analyses as well as into mechanisms of thermal stability. This is the first nucleoside hydrolase from Archaea.

The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis.

Lysins are peptidoglycan hydrolases that are produced by bacteriophage and act to lyse the bacterial host cell wall during progeny phage release. Here, we describe the structure and function of a novel bacteriophage-derived lysin, PlyB, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342. This molecule comprises an N-terminal catalytic domain (PlyB(cat)) and a C-terminal bacterial SH3-like domain, SH3b. It is shown that both domains are required for effective catalytic activity against ATCC 4342. Further, PlyB has specific activity comparable to the phage lysin PlyG, an amidase being developed as a therapeutic against anthrax. In contrast to PlyG, however, the 1.6 A X-ray crystal structure of PlyB(cat) reveals that the catalytic domain adopts the glycosyl hydrolase (GH)-25, rather than phage T7 lysozyme-like fold. PlyB therefore represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.

Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex.

A chitinase isolated from the latex of the tropical species Carica papaya has been purified to homogeneity and crystallized. This enzyme belongs to glycosyl hydrolase family 19 and exhibits exceptional resistance to proteolysis. The initially observed crystals, which diffracted to a resolution of 2.0 A, were improved through modification of the crystallization protocol. Well ordered crystals were subsequently obtained using N-acetyl-D-glucosamine, the monomer resulting from the hydrolysis of chitin, as an additive to the crystallization solution. Here, the characterization of a chitinase crystal that belongs to the monoclinic space group P2(1), with unit-cell parameters a = 69.08, b = 44.79, c = 76.73 A, beta = 95.33 degrees and two molecules per asymmetric unit, is reported. Diffraction data were collected to a resolution of 1.8 A. Structure refinement is currently in progress.

Mono-ADP-Ribosylhydrolase Assays.

Despite substantial progress in ADP-ribosylation research in recent years, the identification of ADP-ribosylated proteins, their ADP-ribose acceptors sites, and the respective writers and erasers remains challenging. The use of recently developed mass spectrometric methods helps to further characterize the ADP-ribosylome and its regulatory enzymes under different conditions and in different cell types. Validation of these findings may be achieved by in vitro assays for the respective enzymes. In the below method, we describe how recombinant ADP-ribosylated proteins are demodified in vitro with mono-ADP-ribosylhydrolases of choice to elucidate substrate and potentially also site specificity of these enzymes.

New nucleoside hydrolase with transribosylation activity from Agromyces sp. MM-1 and its application for enzymatic synthesis of 2'-O-methylribonucleosides.

Microorganisms were screened for transribosylation activity between 2'-O-methyluridine (2'-OMe-UR) and nucleobases, for the purpose of developing a biotransformation process to synthesize 2'-O-methylribonucleosides (2'-OMe-NRs), which are raw materials for nucleic acid drugs. An actinomycete, Agromyces sp. MM-1 was found to produce 2'-O-methyladenosine (2'-OMe-AR) when whole cells were used in a reaction mixture containing 2'-OMe-UR and adenine. The enzyme responsible for the transribosylation was partially purified from Agromyces sp. MM-1 cells through a six-step separation procedure, and identified as a nucleoside hydrolase family enzyme termed AgNH. AgNH was a bi-functional enzyme catalyzing both hydrolysis towards 2'-OMe-NRs and transribosylation between 2'-OMe-UR and various nucleobases as well as adenine. In the hydrolysis reaction, AgNH preferred guanosine analogues as its substrates. In the transribosylation reaction, AgNH showed strong activity towards 6-chloroguanine, with 25-fold relative activity when adenine was used as the acceptor substrate. The transribosylation reaction product from 2'-OMe-UR and 6-chloroguanine was determined to 2'-O-methyl-6-chloroguanosine (2'-OMe-6ClGR). Under the optimal conditions, the maximum molar yield of 2'-OMe-6ClGR reached 2.3% in a 293-h reaction, corresponding to 440 mg/L.

ADP-Ribosyl-Acceptor Hydrolase Activities Catalyzed by the ARH Family of Proteins.

The ARH family of ADP-ribosyl-acceptor hydrolases is composed of three 39-kDa proteins (ARH1, 2, and 3), which hydrolyze specific ADP-ribosylated substrates. ARH1 hydrolyzes mono(ADP-ribosyl)ated arginine, which results from actions of cholera toxin and other nicotinamide adenine dinucleotide (NAD+):arginine ADP-ribosyl-transferases, while ARH3 hydrolyzes poly(ADP-ribose) and O-acetyl-ADP-ribose, resulting from the action of poly(ADP-ribose) polymerases and sirtuins, respectively. ARH2 has not been reported to have enzymatic activity, because of differences in the catalytic domain. Thus, the substrate specificities of ARH1 and ARH3 proteins result in unique cellular functions. In this chapter, we introduce several methods to monitor the activities of the ARH family members.

Invariant Ser211 is involved in the catalysis of PD-L4, type I RIP from Phytolacca dioica leaves.

Multiple sequence alignment analysis of ribosome inactivating proteins (RIPs) has revealed the occurrence of an invariant seryl residue in proximity of the catalytic tryptophan. The involvement of this seryl residue in the catalytic mechanism of RIPs was investigated by site-directed mutagenesis in PD-L4, type 1 RIP isolated from Phytolacca dioica leaves. We show that the replacement of Ser211 with Ala apparently does not influence the N-beta-glycosidase activity on ribosomes (determined as IC(50) in a cell-free system), but it reduces the adenine polynucleotide glycosylase activity (APG), assayed spectrophotometrically on other substrates such as DNA, rRNA, and poly(A). The ability of PD-L4 to deadenylate polynucleotides appears more sensitive to the Ser211Ala replacement when poly(A) is used as substrate, as only 33% activity is retained by the mutant, while with more complex and heterogeneous substrates such as DNA and rRNA, its APG activity is 73% and 66%, respectively. While the mutated protein shows a conserved secondary structure by CD, it also exhibits a remarkably enhanced tryptophan fluorescence. This indicates that, although the overall protein tridimensional structure is maintained, removal of the hydroxyl group locally affects the environment of a Trp residue. Modelling and docking analyses confirm the interaction between Ser211 and Trp207, which is located within the active site, thus affecting RIP adenine polynucleotide glycosylase activity. Data accumulated so far confirm the potential involvement of Ser211 in the catalytic mechanism of type 1 RIP PD-L4 and a possible role in stabilizing the conformation of Trp207 side chain, which participates actively in the protein enzymatic activity.

Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily.

BACKGROUND: Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins. RESULTS: Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G. CONCLUSIONS: S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.