Biochemical Characterization of the Pneumococcal Glucose 1-Phosphate Uridylyltransferase (GalU) Essential for Capsule Biosynthesis

The glucose 1-phosphate uridylyltransferase (GalU) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of Streptococcus pneumoniae. The pneumococcal GalU protein was overexpressed in Escherichia coli, and purified. GalU showed a pI of 4.23, and catalyzed the reversible formation of UDP-glucose and pyrophosphate from UTP and glucose 1-phosphate with K_m values of 0.4 mM for UDP-glucose, 0.26 mM for pyrophosphate, 0.19 mM for glucose 1-phosphate, and 0.24 mM for UTP. GalU has an optimum pH of 8–8.5, and requires Mg²⁺ for activity. Neither ADP-glucose nor TDP-glucose is utilized as substrates in vitro. The purification of GalU represents a fundamental step to provide insights on drug design to control the biosynthesis of the main pneumococcal virulence factor.     

Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci

BackgoundStudying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy.MethodsWe report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (k_cat/K_m) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2.ResultsAll enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules.ConclusionsResults support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications.General significanceResults presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.     

Spinophilin acts as a tumor suppressor by regulating Rb phosphorylation

The scaffold protein Spinophilin (SPN) is a regulatory subunit of phosphatase1a located at 17q21.33. This region is frequently associated with microsatellite instability and LOH containing a relatively high density of known tumor suppressor genes, including BRCA1. Several linkage studies have suggested the existence of an unknown tumor suppressor gene distal to BRCA1. Spn may be this gene, but the mechanism through which this gene makes its contribution to cancer has not been described. In this study, we aimed to determine how loss of Spn may contribute to tumorigenesis. We explored the contribution of SPN to PP1a-mediated Rb regulation. We found that the loss of Spn downregulated PPP1CA and PP1a activity, resulting in a high level of phosphorylated Rb and increased ARF and p53 activity. However, in the absence of p53, reduced levels of SPN enhanced the tumorigenic potential of the cells. Furthermore, the ectopic expression of SPN in human tumor cells greatly reduced cell growth. Taken together, our results demonstrate that the loss of Spn induces a proliferative response by increasing Rb phosphorylation, which, in turn, activates p53, thereby neutralizing the proliferative response. We suggest that Spn may be the tumor suppressor gene located at 17q21.33 acting through Rb regulation.     

The relBE2Spn Toxin-Antitoxin System of Streptococcus pneumoniae: Role in Antibiotic Tolerance and Functional Conservation in Clinical Isolates

Type II (proteic) chromosomal toxin-antitoxin systems (TAS) are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the relBE family is one of the most abundant, being present in the three first sequenced strains of Streptococcus pneumoniae (D39, TIGR4 and R6). To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, ΔrelB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection.     

Two putative glutamate decarboxylases of Streptococcus pneumoniae as possible antigens for the production of anti-GAD65 antibodies leading to type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) has been increasing in prevalence in the last decades and has become a global burden. Autoantibodies against human glutamate decarboxylase (GAD65) are among the first to be detected at the onset of T1DM. Diverse viruses have been proposed to be involved in the triggering of T1DM because of molecular mimicry, i.e., similarity between parts of some viral proteins and one or more epitopes of GAD65. However, the possibility that bacterial proteins might also be responsible for GAD65 mimicry has been seldom investigated. To date, many genomes of Streptococcus pneumoniae (the pneumococcus), a prominent human pathogen particularly prevalent among children and the elderly, have been sequenced. A dataset of more than 9000 pneumococcal genomes was mined and two different (albeit related) genes (gadA and gadB), presumably encoding two glutamate decarboxylases similar to GAD65, were found. The various gadA_Spn alleles were present only in serotype 3 pneumococci belonging to the global lineage GPSC83, although some homologs have also been discovered in two subspecies of Streptococcus constellatus (pharyngis and viborgensis), an isolate of the group B streptococci, and several strains of Lactobacillus delbrueckii. Besides, gadB_Spn alleles are present in > 10% of the isolates in our dataset and represent 16 GPSCs with 123 sequence types and 20 different serotypes. Sequence analyses indicated that gadA- and gadB-like genes have been mobilized among different bacteria either by prophage(s) or by integrative and conjugative element(s), respectively. Substantial similarities appear to exist between the putative pneumococcal glutamate decarboxylases and well-known epitopes of GAD65. In this sense, the use of broader pneumococcal conjugate vaccines such as PCV20 would prevent the majority of serotypes expressing those genes that might potentially contribute to T1DM. These results deserve upcoming studies on the possible involvement of S. pneumoniae in the etiopathogenesis and clinical onset of T1DM.

     

Enhanced UDP-glucose and UDP-galactose by homologous overexpression of UDP-glucose pyrophosphorylase in Lactobacillus casei

UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides.     

Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples

We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ _max=390 nm) is converted to a red product (λ _max=486 nm). Since CA can irreversibly inhibit β-lactamase activity, the level of CA in a sample can be measured as a function of the A ₃₉₀/A ₄₈₆ ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L⁻¹ and 50 μg L⁻¹ to 10 mg L⁻¹, respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.     

Functional Analysis of the Lactococcus lactis galU and galE Genes and Their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis

We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.     

The Cell Division Protein FtsZ from Streptococcus pneumoniae Exhibits a GTPase Activity Delay

The cell division protein FtsZ assembles in vitro by a mechanism of cooperative association dependent on GTP, monovalent cations, and Mg²⁺. We have analyzed the GTPase activity and assembly dynamics of Streptococcus pneumoniae FtsZ (SpnFtsZ). SpnFtsZ assembled in an apparently cooperative process, with a higher critical concentration than values reported for other FtsZ proteins. It sedimented in the presence of GTP as a high molecular mass polymer with a well defined size and tended to form double-stranded filaments in electron microscope preparations. GTPase activity depended on K⁺ and Mg²⁺ and was inhibited by Na⁺. GTP hydrolysis exhibited a delay that included a lag phase followed by a GTP hydrolysis activation step, until reaction reached the GTPase rate. The lag phase was not found in polymer assembly, suggesting a transition from an initial non-GTP-hydrolyzing polymer that switches to a GTP-hydrolyzing polymer, supporting models that explain FtsZ polymer cooperativity.     

Antigenic diversity of cytochromes c3 from the anaerobic,sulfate-reducing bacteria,Desulfovibrio

An indirect enzyme-linked immunoadsorption assay (ELISA) was developed for cytochrome c₃ using antisera to the cytochromes fromDesulfovibrio africanus Benghazi, Desulfovibrio vulgaris Hildenborough andDesulfovibrio salexigens British Guiana. The ELISA system was used to test for cross-reactions between these antisera and the heterologous antigens. In contrast to previous experiments using the Ouchterlony technique, all of the cytochromes c₃ tested exhibited some degree of cross-reaction. Considerable variation was seen in cross-reactions for cytochromes c₃ from differing strains ofD. desulfuricans. This observation raises questions about the taxonomic relatedness of these strains. No cross-reaction was seen with eukaryotic cytochrome c or withD. vulgaris cytochrome c₅₅₃. The data demonstrate that cytochrome c₃ is capable of undergoing nonprecipitating cross-reactions, and thus may not be as immunologically unique as was once thought.Abbreviations ELISA Enzyme-linked immunoadsorption assay     

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10⁴ colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP_i and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP_i-recycling loop was completed using ATP sulfurylase and adenosine 5′ phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.     

HPTLC Autography Based Screening and Isolation of Mushroom Tyrosinase Inhibitors of European Plant Species

In the course of this project, 133 plants were evaluated on their ability to inhibit tyrosinase, a key enzyme in melanogenesis. The screening was performed by means of a HPTLC autographic assay, resulting in the selection of three plants, Asplenium trichomanes, Pinus uncinata, and Scutellaria altissima, with promising tyrosinase inhibiting activities. With the aid of the HPTLC assay, it was not only possible to select the most interesting plant extracts, but also to monitor the activity‐guided fractionation which, in a relatively short time period, led to the isolation of active principles. Benzoic acid, roseoside, and dihydrovomifoliol‐O‐β‐d ‐glucopyranoside could be identified as tyrosinase inhibitors present in P. uncinata. Globularin turned out to be the active principle of S. altissima, and 4‐ethenylphenyl 6‐O‐(6‐deoxy‐α‐l ‐mannopyranosyl)‐β‐d ‐glucopyranoside was detected as tyrosinase inhibitor of A. trichomanes. The pure compounds were tested also in a 96 well‐plate assay in order to determine their IC₅₀ values. The lowest IC₅₀ value (42 μm ) could be obtained for globularin, whereas the other compounds, e. g., benzoic acid exhibited a rather high IC₅₀ value (IC₅₀=552 μm ). This stood in clear contrast to the autographic assay, but is has to be taken into account that the outcome of the autography assay is not only depending on the IC50 value of a compound, but also on the content of the respective constituent in the extract.     

Assay for etoposide in human serum using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

An HPLC assay for etoposide in human serum was developed. Serum, spiked with podophyllotoxin (internal standard), was treated with sodium dodecyl sulphate prior to solid phase extraction. Analysis was performed on a 300×3.9 mm Bondclone 10 C18 column coupled with a fluorometric detector (λ_ex 230 nm, λ_em 330 nm). The retention times for etoposide and podophyllotoxin were 14 and 28 min respectively. The range of assay was 0.5 to 20 μg/ml with a detection limit of 0.2 μg/ml. This assay is suitable for use in clinical studies with etoposide.     

Rat Liver Catechol-O-Methyltransferase Kinetics and Assay Methodology

Abstract

In mammals, catechol-O-methyltransferase (COMT) is distributed throughout various organs, the highest activities being found in the liver and kidney. However, comparisons of the kinetic parameters are difficult to perform, since the experimental procedures in the enzyme assay vary quite considerably. The present work was aimed at studying the optimal liver COMT assay conditions for determining the kinetics of the enzyme. The COMT assay was performed with liver homogenates from 60 days old male Wistar rats with adrenaline (AD) as the substrate. Time course experiments using 100 μM S-adenosyl-L-methionine (SAMe) and 300 μM AD showed linearity of O-methylation reaction upto 10min. Using 100μM SAMe, V_max (nmol mg protein' h^?1) and K_m (μM) values progressively decreased respectively from 22.1 and 104.8 at 5mindown to 5.8 and 24.62 at 60 min incubation periods. This decrease was not due to end-product inhibition. Using 2500 μM AD, K_m values (μM) for the methyl donor SAMe increased progressively from 174 at 5 min upto 1192.5 at 60 min; upto 30 min of incubation F_max values did not change. When a 5 min incubation period and 500 μM SAMe were used, V_max and K_m values for liver COMT were 63.4 nmol mg protein^?1h^?1 and 261.1 μM, respectively. It is concluded that an incubation period of 5 min and a SAMe concentration of 500 μM provide optimal conditions for the liver homogenate COMT assay.     

A Fluorescence-Based Homogeneous Assay for Measuring Activity of UDP–3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase

UDP–3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP–3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogenous fluorescence-based assay was developed that uses UDP–3-O-(N-hexyl-propionamide)-N-acetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP–GlcNAc by enzymatic conversion to UDP–MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K_m of 367 μM and k_cat of 0.36 s⁻¹, compared to 2 μM and 1.5 s⁻¹ for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC₅₀s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.     

Gamma-glutamyl compounds: substrate specificity of gamma-glutamyl transpeptidase enzymes

Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites, and neuroactive compounds. Two cell surface enzymes that metabolize gamma-glutamyl compounds have been identified: gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetic analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative, and conducted at physiological pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The K_m value for reduced glutathione was 11 μM for both GGT1 and GGT5. However, the K_m values for oxidized glutathione were 9 μM for GGT1 and 43 μM for GGT5. Our data show that the K_m values for leukotriene C₄ are equivalent for GGT1 and GGT5 at 10.8 and 10.2 μM, respectively. This assay was also used to evaluate serine–borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than in inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism, and other pathways that involve gamma-glutamyl compounds.