Search

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) enzyme in psychrophilic bacteria has gradually attracted the attention of researchers. A novel gene, deoC (681 bp), encoding DERA Psy , was identified in Pseudomonas syringae pv. syringae B728a, recombinantly expressed in E. coli BL21 and purified via affinity chromatography, which yielded a homodimeric enzyme of 23 kDa. The specific activity of DERA Psy toward 2-deoxy-d-ribose-5-phosphate (DR5P) was 7.37 ± 0.03 U/mg, and 61.32% of its initial activity remained after incubation in 300 mM acetaldehyde at 25 °C for 2 h. Based on the calculation results (dock binding free energy) with the ligand chloroacetaldehyde (CAH), five target substitutions (T16L, F69R, V66K, S188V, and G189R) were identified, in which the DERA Psy mutant (G189R) exhibited higher catalytic activity toward DR5P than DERA Psy . Only the DERA Psy mutant (V66K) exhibited 12% higher activity toward chloroacetaldehyde and acetaldehyde condensation reactions than DERA Psy . Fortunately, the aldehyde tolerance of these mutants exhibited no significant decline compared with the wild type. These results indicate an effective strategy for enhancing DERA activity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1,6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z' (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.

Background: Several refugee settlements in Bangladesh have provided housing and medical care for the forcibly-displaced Myanmar nationals (FDMN, also known as Rohingya) population. The identification of malaria infection status in the refugee settlements is useful in treating infected persons and in developing malaria prevention recommendations. Assays for Plasmodium antigens and human IgG against Plasmodium parasites can be used as indicators to determine malaria infection status and exposure., Methods: Dried blood spot (DBS) samples (N = 1239) from a household survey performed April-May 2018 in three settlements in Cox's Bazar district, Bangladesh were utilized for a sample population of children from ages 1-14 years of age. The samples were tested using a bead-based multiplex antigen assay for presence of the pan-Plasmodium antigen aldolase as well as Plasmodium falciparum histidine rich protein 2 (HRP2). A bead-based multiplex assay was also used to measure human IgG antibody response to P. falciparum, Plasmodium malariae, and Plasmodium vivax merozoite surface protein 1 antigen (MSP1) isoforms, and P. falciparum antigens LSA1, CSP, and GLURP-R0., Results: There were no detectable Plasmodium antigens in any samples, suggesting no active malaria parasite infections in the tested children. IgG seroprevalence was highest to P. vivax (3.1%), but this was not significantly different from the percentages of children antibody responses to P. falciparum (2.1%) and P. malariae (1.8%). The likelihood of an anti-Plasmodium IgG response increased with age for all three malaria species. Evidence of exposure to any malaria species was highest for children residing 8-10 months in the settlements, and was lower for children arriving before and after this period of time., Conclusions: Absence of Plasmodium antigen in this population provides evidence that children in these three Bangladeshi refugee settlements did not have malaria at time of sampling. Higher rates of anti-malarial IgG carriage from children who were leaving Myanmar during the malaria high-transmission season indicate these migrant populations were likely at increased risk of malaria exposure during their transit.

The impact of malaria on global health has continually prompted the need to develop more effective diagnostic strategies that could overcome deficiencies in accurate and early detection. In this review, we examine the various biosensor-based methods for malaria diagnostic biomarkers, namely; Plasmodium falciparum histidine-rich protein 2 (PfHRP-2), parasite lactate dehydrogenase (pLDH), aldolase, glutamate dehydrogenase (GDH), and the biocrystal hemozoin. The models that demonstrate a potential for field application have been discussed, looking at the fabrication and analytical performance characteristics, including (but not exclusively limited to): response time, sensitivity, detection limit, linear range, and storage stability, which are first summarized in a tabular form and then described in detail. The conclusion summarizes the state-of-the-art technologies applied in the field, the current challenges and the emerging prospects for malaria biosensors.

Fructose-1,6-bisphosphate aldolase (FBA) is a key metabolic enzyme, which is involved in glycolysis, gluconeogenesis and the Calvin cycle. The distinct physiological roles of FBAs in various organisms have been reported; however, in cyanobacteria, the functional characterization of FBAs and investigation of the intracellular dynamics of FBAs largely remains unknown. Here, we utilized a two-step chromatographic technique to identify a class I FBA (CI-FBA), which we named H2846. H2846 was induced by salt stress in the halotolerant cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece 7418). Phylogenetic analysis showed that H2846-like CI-FBAs existed mainly in cyanobacterial species that inhabit hypersaline environments. Subcellular fractionation revealed that H2846 localized in the cytosolic and periplasmic spaces and size-exclusion chromatography suggested that H2846 formed a homohexamer. The CI-FBA activity of recombinant H2846-mediated cleavage of fructose bisphosphate (FBP) was characterized using a coupled enzymatic assay. This analysis allowed us to determine the K m and V max values of recombinant H2846, which were then compared to previously reported K m and V max values of several FBAs. Our data suggested that H2846 was likely responsible for the salt stress-induced CI-FBA activity from the total soluble protein extracts derived from Halothece 7418 cells. Moreover, heterologous expression of H2846 but not H2847, a class II FBA (CII-FBA), conferred salt stress tolerance to the salt-sensitive freshwater cyanobacterium, Synechococcus elongatus PCC 7942, which only contains the CII-FBA, S1443. S. elongatus PCC 7942 with a S1443 gene deletion was complemented by H2847 expression, but was not complemented by expression of H2846. Taken together, these results indicate the functional differences between two distinct sets of FBAs in cyanobacteria. H2846 is an active CI-FBA that contributes to the mechanism of salt stress tolerance in Halothece 7418., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Extraction of protein from macroalgae, currently defined as "novel food", is challenging and limited information about the health impacts of these proteins is available. Here, we report on a non-thermal, chemical-free green macroalgae Ulva sp. protein extraction by osmotic shock combined with pulsed electric fields (PEF) followed by hydraulic pressure. The extracted proteins were identified and annotated to allergens using sequence similarity. The allergenicity potential of PEF extracted proteins was compared to osmotic shock extracts and complete Ulva sp. proteome, extracted with the thermochemical method. The PEF extracts contained 'superoxide dismutase' (SOD), a known food allergen, osmotic shock extract contained 'troponin C', and thermochemical extract contained two additional potential food allergens 'aldolase A' and 'thioredoxin h'. This study shows an importance and the need for deep investigation of algal proteins and protein extraction technology health impacts prior to large-scale release to the market of "novel food" derived proteins., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting Plasmodial histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2 is only expressed by Plasmodium falciparum parasites and the protein is not expressed in several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P. falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) Escherischia coli host cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV) specific to P. falciparum GAPDH and one common to all mammalian malaria species (CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two recombinant proteins and the three epitopes and affinity purified. The antibodies detected the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting potential as a malaria diagnostic biomarker., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum., Methods: FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR., Results: SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum., Conclusion: SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.

Fructose-1,6-bisphosphate aldolase is one of the most important enzymes in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The full-length fbaB gene encoding fructose-1,6-bisphosphate aldolase class I (FBPA I) was cloned from Escherichia coli strain BL21. FBPA I was overexpressed in E. coli and purified. Biochemical analysis found that the optimum reaction temperature of FBPA I is 330.5 K and that the enzyme has a high temperature tolerance. Crystals of recombinant FBPA I were obtained by the sitting-drop vapour-diffusion technique in a condition consisting of 19 mg ml(-1) FBPA I in 0.1 M Tris pH 9.0, 10%(w/v) polyethylene glycol 8000 and diffracted to 2.0 Å resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 217.7, b = 114.9, c = 183.9 Å, β = 124.6°. The asymmetric unit of these crystals may contain ten molecules, giving a Matthews coefficient of 2.48 Å(3) Da(-1) and a solvent content of 50.5%.

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9) mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.

By the action of D-fructose 1,6-bisphosphate aldolases (FruA) from rabbit muscle and Staphylococcus carnosus, various ketoses were synthesized from glyceraldehydes or other aliphatic aldehydes as acceptors in a one-pot four-enzyme system., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

4-Hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL), an enzyme used in the synthesis of (2S,3R,4S)-4-hydroxyisoleucine, was crystallized in the absence and the presence of 2-ketobutyrate as one of its substrates by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. Crystals of asHPAL grown without and with 2-ketobutyrate diffracted to 1.60 and 1.55 Å resolution and belonged to space group C2, with unit-cell parameters a = 116.8, b = 88.2, c = 85.3 Å, β = 122.3° and a = 116.2, b = 88.1, c = 85.0 Å, β = 122.3°, respectively.

Background: Candida albicans is an opportunistic dimorphic fungus commonly present in the human oral cavity that causes infections in immunocompromised patients. The antigen variability, influenced by growth conditions, is a pathogenicity factor., Aims: To determine the effect of nutritional and heat stress on the antigen expression of C. albicans, and to identify major antigens recognized by human salivary secretory immunoglobulin A (sIgA)., Methods: Under various different nutritional conditions, heat shock was induced in C. albicans cells in stationary and exponential growth phases. The expression of protein determinants of C. albicans was assessed by Western blot analysis against human saliva. The antigens were purified and characterized by two-dimensional electrophoresis and identified by protein microsequencing., Results: Five antigens recognized by salivary IgA were characterized as mannoproteins due to their reactivity with concanavalin A. They did not show reactivity with anti-heat shock protein monoclonal antibodies. Two of them (42 and 36 kDa) were found to be regulated by heat shock and by nutritional stress and they were identified as phosphoglycerate kinase and fructose bisphosphate aldolase, respectively., Conclusions: These glycolytic enzymes are major antigens of C. albicans, and their differential expression and recognition by the mucosal immune response system could be involved in protection against oral infection., (Copyright © 2011 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Nickel is toxic to all forms of life, but the mechanisms of cell damage are unknown. Indeed, environmentally relevant nickel levels (8 µM) inhibit wild-type Escherichia coli growth on glucose minimal medium. The same concentration of nickel also inhibits growth on fructose, but not succinate, lactate or glycerol; these results suggest that fructose-1,6-bisphosphate aldolase (FbaA) is a target of nickel toxicity. Cells stressed by 8 µM Ni(II) for 20 min lost 75% of their FbaA activity, demonstrating that FbaA is inactivated during nickel stress. Furthermore, overexpression of fbaA restored growth of an rcnA mutant in glucose minimal medium supplemented with 4 µM Ni(II), thus confirming that FbaA is a primary target of nickel toxicity. This class II aldolase has an active site zinc and a non-catalytic zinc nearby. Purified FbaA lost 80 % of its activity within 2 min when challenged with 8 µM Ni(II). Nickel-challenged FbaA lost 0.8 zinc and gained 0.8 nickel per inactivated monomer. FbaA mutants (D144A and E174A) affecting the non-catalytic zinc were resistant to nickel inhibition. These results define the primary site of nickel toxicity in E. coli as the class II aldolase FbaA through binding to the non-catalytic zinc site., (© 2011 Blackwell Publishing Ltd.)

Fructose 1,6-bisphosphate (FBP) aldolase has been used as biocatalyst in the synthesis of several pharmaceutical compounds such as monosaccharides and analogs. Is has been suggested that microbial metal-dependant Class II aldolases could be better industrial catalysts than mammalian Class I enzyme because of their greater stability. The Class II aldolases from four microbes were subcloned into the Escherichia coli vector pT7-7, expressed and purified to near homogeneity. The kinetic parameters, temperature stability, pH profile, and tolerance to organic solvents of the Class II enzymes were determined, and compared with the properties of the Class I aldolase from rabbit muscle. Contrary to results obtained previously with the E. coli Class II aldolase, which was reported to be more stable than the mammalian enzyme, other recombinant Class II aldolases were found to be generally less stable than the Class I enzyme, especially in the presence of organic solvents. Class II aldolase from Bacillus cereus showed higher temperature stability than the other enzymes tested, but only the Mycobacterium tuberculosis Class II aldolase had a stability comparable to the Class I mammalian enzyme under assay conditions. The turnover number of the recombinant M. tuberculosis and Magnaporthe grisea Class II type A aldolases was comparable or higher than that of the Class I enzyme. The recombinant B. cereus and Pseudomonas aeruginosa Class II type B aldolases had very low turnover numbers and low metal content, indicating that the E. coli overexpression system may not be suitable for the Class II type B aldolases from these microorganisms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co2+ and inhibited by EDTA. The enzyme displays low K(m) to fructose-1,6-bisphosphate (FBP) and higher K(m) to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co2+ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C(1) assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.

The pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of [1-(13)C]- and [3-(13)C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type pathway in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene, kdgA, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctional KD(P)GA as a key enzyme.

Background: The standard in vitro test to assess anti-malarial activity of chemical compounds is the [3H]hypoxanthine incorporation assay. It is a radioactivity-based method to measure DNA replication of Plasmodium in red blood cells. The method is highly reproducible, however, the handling of radioactive material is costly, hazardous and requires the availability of appropriate technology and trained staff. Several other ways to evaluate in vitro anti-malarial activity do exist, all with their own assets and limitations., Methods: The newly developed double-antibody sandwich ELISA described here is based on the properties of a non-overlapping pair of monoclonal antibodies directed against Plasmodium falciparum aldolase. This glycolytic enzyme possesses some unique nucleotide sequences compared to the human isoenzymes and has been highly conserved through evolution. Out of twenty possibilities, the most sensitive antibody pair was selected and used to quantitatively detect parasite aldolase in infected blood lysates., Results: A total of 34 compounds with anti-malarial activity were tested side-by-side by ELISA and the [3H]hypoxanthine incorporation assay. The novel ELISA provided IC 50s closely paralleling those from the radioactivity-based assay (R = 0.99, p < 0.001). At the investigated assay conditions (72 h incubation time, parasitaemia = 0.3%), the assay was found to be reproducible and easy to perform., Conclusion: The newly developed ELISA presents several advantages over the comparative method, the [3H]hypoxanthine incorporation assay. The assay is highly reproducible, less hazardous (involves no radioactivity) and requires little and cheap technical equipment. Relatively unskilled personnel can conduct this user-friendly assay. All this makes it attractive to be employed in resource-poor laboratories.

By screening microorganisms that are capable of assimilating alpha-methyl-DL-serine, we detected alpha-methylserine aldolase in Ralstonia sp. strain AJ110405, Variovorax paradoxus AJ110406, and Bosea sp. strain AJ110407. A homogeneous form of this enzyme was purified from Ralstonia sp. strain AJ110405, and the gene encoding the enzyme was cloned and expressed in Escherichia coli. The enzyme appeared to be a homodimer consisting of identical subunits, and its molecular mass was found to be 47 kDa. It contained 0.7 to 0.8 mol of pyridoxal 5'-phosphate per mol of subunit and could catalyze the interconversion of alpha-methyl-L-serine to L-alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from alpha-methyl-L-serine but not from alpha-methyl-D-serine, L-serine, or D-serine. Alpha-methyl-L-serine synthesis activity was detected when L-alanine was used as the substrate. In contrast, no activity was detected when D-alanine was used as the substrate. In the alpha-methyl-L-serine synthesis reaction, the enzymatic activity was inhibited by an excess amount of formaldehyde, which was one of the substrates. We used cells of E. coli as a whole-cell catalyst to express the gene encoding alpha-methylserine aldolase and effectively obtained a high yield of optically pure alpha-methyl-L-serine using L-alanine and formaldehyde.

Fructose-1,6-bisphosphate aldolase (FBPA) is an ubiquitous enzyme essential for glycolysis, gluconeogenesis and the Calvin cycle. It has been demonstrated to induce immune responses and to be useful in the immunodiagnosis of malaria. In this study, FBPA was cloned from the adult worms of Schistosoma japonicum and tested as an antigen for the diagnosis of S. japonicum infection in water buffaloes. Enzyme-linked immunosorbent assay (ELISA) was performed on the sera from 32 infected water buffaloes and 20 negative controls using the recombinant FBPA protein or soluble worm antigen preparation (SWAP) as an antigen. The OD cut-off values were determined to be 0.57 with 100% specificity and 100% sensitivity for the FBPA ELISA and 1.13 with 93.8% specificity and 95.0% sensitivity for the SWAP ELISA. These findings indicate that the recombinant FBPA of S. japonicum should be an useful diagnostic tool for the detection of antibodies against S. japonicum.

The alpha-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from alpha-methyl-L-serine was detected in the cell-free extract of E.coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The Vmax and Km of the enzyme for the formaldehyde release reaction from alpha-methyl-L-serine were 1.89 micromol min(-1) mg(-1) and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of alpha-methyl-L-serine and alpha-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5'-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.

Antigenic proteins of Echinostoma caproni (Trematoda) against mouse IgM, IgA, IgG, IgG1 and IgG2a were investigated by immunoproteomics. Excretory/secretory products (ESP) of E. caproni separated by two-dimensional (2D) gel electrophoresis were transferred to nitrocellulose membranes and probed with the different mouse immunoglobulin classes. A total of four proteins (enolase, 70 kDa heat-shock protein (HSP-70), actin and aldolase) were accurately identified. Enolase was recognized in eight different spots of which seven of them were detected in the expected molecular weight and were recognized by IgA, IgG or IgG and IgG1. Another spot identified as enolase at 72 kDa was only recognized by IgM. Digestion with N-glycosidase F of the 72 kDa band rendered a polypeptide with an apparent molecular weight similar to that expected for enolase recognized by Western immunoblotting using anti-enolase antibodies. This suggests that glycosylated forms of enolase may be involved in the early thymus-independent responses against E. caproni. Early IgM responses were also generated by actin and the HSP-70 which suggests that these proteins are exposed early to the host and may be of importance in the parasite establishment. The IgA responses also appear to be mediated by the HSP-70 and aldolase which could be related with the close contact of these proteins with the host mucosal surface after secretion.

Previously we have shown that aldolase (ALD; EC 4.1.2.13) is present in cardiomyocyte nuclei. Now, we focused our attention on ALD localization in smooth muscle cells. Immunocytochemical methods were used to study the subcellular localization of ALD. Aldolase was localized in the cytoplasm as well as in the nuclei. Within the nuclei ALD was located in the heterochromatin region. Native polyacrylamide gel electrophoresis followed by aldolase activity staining in gel was used to study the ALD isoenzyme pattern in porcine smooth muscle cells. Two ALD isoenzymes, A and C, were found in these cells but in the nuclei only the muscle isoenzyme was detected. To support the nuclear localization of ALD, measurement of aldolase activity in the smooth muscle cell nuclei isolated from porcine stomach was performed. The ALD activity in the isolated nuclei was detectable only after preincubation of the nuclear fraction with Triton X-100 and high concentration of KCl.

The macrophage protein tyrosine phosphatase-1 SHP-1 has been implicated in the pathogenesis of infection with leishmania. To identify the factors that may interact with SHP-1, Leishmania donovani promastigote lysates were added to a GST-SHP-1 affinity matrix. A 44kDa specifically bound protein was identified as leishmania fructose-1,6-bisphosphate aldolase (aldolase). Purified leishmania aldolase bound to SHP-1 indicating that the interaction was direct. In contrast, purified mammalian aldolase did not bind to SHP-1. Consistent with this, leishmania aldolase activated SHP-1 in vitro, whereas mammalian aldolase did not. The presence of leishmania aldolase in the cytosolic fractions prepared from infected macrophages indicated that leishmania aldolase is exported from phagolysosomes in infected cells where it can target host cytosolic proteins. In fact, co-immunoprecipitation showed association of leishmania aldolase with SHP-1. Moreover, leishmania aldolase-expressing macrophages showed the deactivated phenotype of leishmania infected cells as judged by much reduced inability to induce expression of nitric-oxide synthase in response to interferon-gamma treatment. Collectively, these data show that leishmania aldolase is a novel SHP-1 binding and activating protein that contributes to macrophage dysfunction.

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.

A two-step enzymatic synthesis process of 4-hydroxyisoleucine is suggested. In the first step, the aldol condensation of acetaldehyde and alpha-ketobutyrate catalyzed by specific aldolase results in the formation of 4-hydroxy-3-methyl-2-keto-pentanoate (HMKP). In the second step, amination of HMKP by the branched-chain amino acid aminotransferase leads to synthesis of 4-hydroxyisoleucine. An enzyme possessing HMKP aldolase activity (asHPAL) was purified 2500-fold from a crude extract of Arthrobacter simplex strain AKU 626. Sequencing of the asHPAL structural gene showed that the purified enzyme belongs to the HpcH/HpaI aldolase family. The 4-hydroxyisoleucine was synthesized in vitro from acetaldehyde, alpha-ketobutyrate and l-glutamate using a coupled aldolase/branched-chain amino acid aminotransferase bienzymatic reaction.

The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.

A soluble fructose-1,6-bisphosphate aldolase enzyme has been purified 50.2-fold (2.36%) at the homogeneity from the electric organ of Electrophorus electricus by one step of DEAE-52 anion exchange chromatography followed by Superose-12 gel filtration-FPLC. Like other aldolase enzymes the E. electricus protein is a dimer with two identical subunits of 45 kDa. The N-terminal (20 residues) revealed a high homology with S. aurata (75%, goldfish), R. ratus and M. musculus (mouse, 80%) enzymes.

Objective: To identify the recombinant aldolase (ALD) of Plasmodium falciparum, and to develop monoclonal antibodies (McAbs) against the recombinant ALD., Methods: ALD gene was amplified by PCR from genomic DNA of FCC1/HN strain, and expressed in E. coli DH5 alpha. BALB/c mice were immunized with the recombinant ALD of P. falciparum via celiac injection for 3 times with 2 weeks interval. Three days after a booster injection, spleen cells of the immunized mice were used for producing McAbs. The immune serum was tested by IFAT and Western blotting., Results: BALB/c mice immunized with purified aldolase protein developed strong immune response to the antigen, and the titer of specific antibody reached 1:10(5) in all immune sera after the third immunization. Moreover, immune sera specifically recognized the cultured P. falciparum. Western blotting showed that the immune sera recognized specifically a Mr 41000 band of crude malaria antigen. No cross-reaction with human red cells was detected. Seven positive hybridoma cell lines were obtained after 3 rows of selection. All the McAbs' subclasses belong to IgG1. IFAT showed that only 4 McAbs could recognize the cultured P. falciparum., Conclusion: Plasmodial aldolase has been successfully expressed and purified, and the established hybridoma cell lines can secrete McAbs specific to the aldolase of P. falciparum.

One of the early steps in the biosynthesis of coenzyme F(420) in Methanocaldococcus jannaschii requires generation of 2-phospho-L-lactate, which is formed by the phosphorylation of L-lactate. Preliminary studies had shown that L-lactate in M. jannaschii is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that L-lactate is formed by the NAD(+)-dependent oxidation of l-lactaldehyde by the MJ1411 gene product. The lactaldehyde, in turn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-1-phosphate by fuculose-1-phosphate aldolase, the MJ1418 gene product.

Both full-length and subgenomic negative-strand RNAs are initiated at the 3' terminus of the positive-strand genomic RNA of the arterivirus, simian hemorrhagic fever virus (SHFV). The SHFV 3'(+) non-coding region (NCR) is 76 nts in length and forms a stem loop (SL) structure that was confirmed by ribonuclease structure probing. Two cell proteins, p56 and p42, bound specifically to a probe consisting of the SHFV 3'(+)NCR RNA. The 3'(+)NCR RNAs of two additional members of the arterivirus genus specifically interacted with two cell proteins of the same size. p56 was identified as polypyrimidine tract-binding protein (PTB) and p42 was identified as fructose bisphosphate aldolase A. PTB binding sites were mapped to a terminal loop and to a bulged region of the SHFV 3'SL structure. Deletion of either of the PTB binding sites in the viral RNA significantly reduced PTB binding activity, suggesting that both sites are required for efficient binding of this protein. Changes in the top portion of the SHFV 3'SL structure eliminated aldolase binding, suggesting that the binding site for this protein is located near the top of the SL. These cell proteins may play roles in regulating the functions of the genomic 3' NCR.

In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.

In classical proteomic studies, the searches in protein databases lead mostly to the identification of protein functions by homology due to the non-exhaustiveness of the protein databases. The quality of the identification depends on the studied organism, its complexity and its representation in the protein databases. Nevertheless, this basic function identification is insufficient for certain applications namely for the development of RNA-based gene-silencing strategies, commonly termed RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants, that require an unambiguous identification of the targeted gene sequence. A PTGS strategy was considered in the study of the infection of Oryza sativa by the Rice Yellow Mottle Virus (RYMV). It is suspected that the RYMV recruits host proteins after its entry into plant cells to form a complex facilitating virus multiplication and spreading. The protein partners of this complex were identified by a classical proteomic approach, nano liquid chromatography tandem mass spectrometry. Among the identified proteins, several were retained for a PTGS strategy. Nevertheless most of the protein candidates appear to be members of multigenic families for which all paralog genes are not present in protein databases. Thus the identification of the real expressed paralog gene with classical protein database searches is impossible. Consequently, as the genome contains all genes and thus all paralog genes, a whole genome search strategy was developed to determine the specific expressed paralog gene. With this approach, the identification of peptides matching only a single gene, called discriminant peptides, allows definitive proof of the expression of this identified gene. This strategy has several requirements: (i) a genome completely sequenced and accessible; (ii) high protein sequence coverage. In the present work, through three examples, we report and validate for the first time a genome database search strategy to specifically identify paralog genes belonging to multigenic families expressed under specific conditions.

A proteomic approach was used to identify a 39 kDa antigen of Paracoccidioides brasiliensis. Amino acid sequences of the N-terminal and of endoproteinase Lys-C digested peptides revealed the protein to be a fructose 1,6-biphosphate aldolase (FBA) Class II of P. brasiliensis. Two cDNA homologues, Pbfba1 and Pbfba2, were cloned and characterized. Pbfba1 encoded a predicted polypeptide of 360 amino acids that was highly homologous in the primary structure to the same enzyme from fungi and bacteria. The other DNA, Pbfba2, encoded a polypeptide predicted to be 363 amino acids. The sequence of Pbfba2 differed significantly from Pbfba1. Phylogenetic and molecular analysis supports the concept of gene duplication for FBAs in P. brasiliensis, constituting a two-member family. Expression analysis demonstrated differential expression for both fbas genes in P. brasiliensis cells.

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.

The Class II fructose 1,6-bisphosphate aldolase (fda, Rv0363c) from the pathogen Mycobacterium tuberculosis H37RV was subcloned in the Escherichia coli vector pT7-7 and purified to near homogeneity. The specific activity (35 U/mg) is approximately 9 times higher than previously reported for the enzyme partially purified from the pathogen. Attempts to express the enzyme with an N-terminal fusion tag yielded inactive, mostly insoluble protein. The native recombinant enzyme is zinc-dependent and has a catalytic efficiency for fructose 1,6-bisphosphate cleavage higher than most Class II aldolases characterized to date. The aldolase has a Km of 20 microM, a kcat of 21 s(-1), and a pH optimum of 7.8. The molecular mass of the enzyme subunits as determined by mass spectrometry is in agreement with the mass calculated on the basis of its gene sequence minus the terminal methionine, 36,413 Da. The enzyme is a homotetramer and retains only two zinc ions per tetramer when transferred to a metal-free buffer, as determined by ICP-MS and by a colorimetric assay using 4-(2-pyridylazo)-resorcinol (PAR) as a chelator. The E. coli expression system reported in this study will facilitate the further characterization of this enzyme and the screening for potential inhibitors.

Cloning and transcriptional regulation of the KlFBA1 gene that codes for the class II fructose-1,6-bisphosphate aldolase of the yeast Kluyveromyces lactis are described. KlFBA1 mRNA diminishes transiently during the shift from hypoxic to fully aerobic conditions and increases in the reversal shift. This regulation is mediated by heme since expression was higher in a mutant defective in heme biosynthesis. KlFBA1 transcription is not induced by calcium-shortage, low temperature, or at stationary phase. These data suggest that KlFBA1 plays a role in the balance between oxidative and fermentative metabolism and that this gene is differentially regulated in K. lactis and Saccharomyces cerevisiae, i.e., a respiratory vs. fermentative yeast.

Ion mobility mass spectrometry (IM-MS) peptide mass mapping experiments were performed using a variety of drift gases (He, N2, Ar and CH4). The drift gases studied cover a range of polarizabilities ((0.2-2.6) x 10(-24) cm3) and the peak capacities obtained for tryptic peptides in each gas are compared. Although the different gases exhibit similar peak capacities (5430 (Ar) to 7580 (N2)) in some cases separation selectivity presumably based on peptide conformers (or conformer populations), is observed. For example the drift time profiles observed for some tryptic peptide ions from aldolase (rabbit muscle) show a dependence on drift gas. The transmission of high-mass ions (m/z > 2000) is also influenced by increased scattering cross-section of the more massive drift gases. Consequently the practical peak capacity for IM-MS separation cannot be assumed to be solely a function of resolution and the ability of a gas to distribute signals in two-dimensional space; rather, peak capacity estimates must account for the transmission losses experienced for peptide ions as the drift gas mass increases., (Copyright 2004 John Wiley & Sons, Ltd.)

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) is strongly inhibited by AMP in vitro and, therefore, at physiological concentrations of substrate and AMP, FBPase should be completely inhibited. Desensitization of rabbit muscle FBPase against AMP inhibition was previously observed in the presence of rabbit muscle aldolase. In this study, we analysed the kinetics of an FBPase catalyzed reaction and interaction between chicken muscle FBPase and chicken muscle aldolase. The initial rate of FBPase reaction vs. substrate concentration shows a maximum activity at a concentration of 20 microM Fru-1,6P2 and then decreases. Assuming rapid equilibrium kinetics, the enzyme-catalyzed reaction was described by the substrate inhibition model, with Ks approximately 5 microM and Ksi approximately 39 microM and factor beta approximately 0.2, describing change in the rate constant (k) of product formation from the ES and ESSi complexes. Based on ultracentrifugation studies, aldolase and FBPase form a hetero-complex with approximately 1:1 stoichiometry with a dissociation constant (Kd) of 3.8 microM. The FBPase-aldolase interaction was confirmed via fluorescence investigation. The aldolase-FBPase interaction results in aldolase fluorescence quenching and its maximum emission spectrum shifting from 344 to 356 nm. The Kd of the FBPase-aldolase complex, determined on the basis of fluorescence changes, is 0.4 microM at 25 degrees C with almost 1:1 stoichiometry. This interaction increases the I(0.5) for the AMP inhibition of FBPase threefold, and slightly affects FBPase affinity to magnesium ions, increasing the Ka and Hill coefficient (n). No effect of aldolase on the FBPase pH optimum was observed. Thus, the decrease in FBPase sensitivity to AMP inhibition enables FBPase to function in vivo thanks to aldolase.

In this study, we have crystallized class II fructose-1,6-bisphosphate aldolase (FBA) from Thermus caldophilus (Tca). Purified Tca FBA is a tetrameric enzyme of 305 residues, which crystallizes in the space group P2(1)2(1)2(1) (cell dimensions a = 98.9, b = 113.1, c = 115.7 A), with four molecules in the asymmetric unit. A complete diffraction data set was obtained from orthorhombic crystals at resolution of 2.2 A.

Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase- and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of (13)C-glucose to (13)C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.

We have developed a novel nickel-silica matrix for the generation of magnetic beads for metal-ion affinity chromatography. In contrast to magnetic Ni-NTA agarose beads, the novel particle type (SiMAC) consists of a magnetic core and a nickel-silica composite matrix with the nickel ions tightly integrated in the silica. This results in a much higher number of chelating groups compared with Ni-NTA agarose beads. With the SiMAC beads, greatly improved purification of histidine-tagged proteins from crude bacterial extracts was achieved. The yield was at least twice as high as with conventional materials, the method is faster, since the coupling step is omitted and there is no need for handling toxic Ni(2+) salts.

Real-time interaction analysis, using the BIAcore biosensor, of rabbit muscle FBPase-aldolase complex revealed apparent binding constant [K(Aapp)] values of about 4.4x10(8) M(-1). The stability of the complex was down-regulated by the glycolytic intermediates dihydroxyacetone phosphate and fructose 6-phosphate, and by the regulator of glycolysis and glyconeogenesis--fructose 2,6-bisphosphate. FBPase in a complex with aldolase was entirely insensitive to inhibition by physiological concentrations of AMP (I(0.5) was 1.35 mM) and the cooperativity of the inhibition was not observed. The existence of an FBPase-aldolase complex that is insensitive to AMP inhibition explains the possibility of glycogen synthesis from carbohydrate precursors in vertebrates' myocytes.

A novel enzyme, D-3-hydroxyaspartate aldolase (D-HAA), catalyzing the conversion of D-3-hydroxyaspartate to glyoxylate plus glycine, was purified to homogeneity from Paracoccus denitrificans IFO 13301. D-HAA is strictly D-specific as to the alpha-position, whereas the enzyme does not distinguish between threo and erythro forms at the beta-position. In addition to D-3-hydroxyaspartate, the enzyme also acts on d-threonine, D-3-3,4-dihydroxyphenylserine, D-3-3,4-methylenedioxyphenylserine, and D-3-phenylserine. The D-HAA gene was cloned and sequenced. The gene contains an open reading frame consisting of 1,161 nucleotides corresponding to 387 amino acid residues. The predicted amino acid sequence displayed 35% and 22% identity with that of the D-threonine aldolase of Arthrobacter sp. DK-38 and Alcaligenes xylosoxidan IFO 12669, respectively. This is the first paper reporting both a purified enzyme with D-3-hydroxyaspartate aldolase activity and also its gene cloning.

Previously determined crystal structures of the zinc enzyme Escherichia coli class II fructose-1,6-bisphosphate aldolase display good agreement for the protein structure but a differing metal-ion organization in the active site. The structure of the enzyme with Cd(2+) in place of Zn(2+) has now been determined to 2.0 A resolution to facilitate cation identification. The protein structure was essentially identical to other structures and five Cd(2+) positions were identified. Two of the cations are at the active site; one corresponds to the catalytic ion and the other provides a structural contribution. These Cd(2+) sites are equivalent to two Zn(2+) ions observed when the enzyme is complexed with a transition-state mimic and confirm our assignment of the roles played by these ions.

The whole genome sequence database for Synechocystis sp. PCC 6803 has revealed the presence of genes encoding class-I (CI) and class-II (CII) fructose-1,6-bisphosphate aldolases (FBAs) in this organism. Two types of FBA from Synechocystis sp. PCC 6803 were separated by chromatography on phenyl-Sepharose. The activity of the enzyme in the major peak was inhibited by the presence of 25 mM EDTA; however, the activity in the minor peak was not. Therefore, the FBA in the former fractions was designated as CII-FBA, and in the latter designated as CI-FBA. CI-FBA was functionally redundant in Synechocystis sp. PCC 6803, while no disruptant for the gene encoding CII-FBA was obtained under photoautotrophic conditions. The kinetic parameters of CI- and CII-FBAs purified from Synechocystis sp. PCC 6803 in the cleavage reaction of FBP were generally similar, except in their reactivity for SBP. The SBP/FBP activity ratio of the CII-FBA was two times higher than that of the CI-FBA.