Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli

Irreversible inactivation of α-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K_D) of 6 ± 1 and 7 ± 1 μM, respectively, ∼6-fold tighter than plasma HCII, with K_D 40 ± 4 μM. Binding of recombinant and deglycosylated HCII to DS, both with K_D 4 ± 1 μM, was ∼4-fold tighter than for plasma HCII, with K_D 15 ± 4 μM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated α-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn¹⁶⁹N-glycan with the HCII heparin-binding site.     

Stability and reactivity of liposome‐encapsulated formate dehydrogenase and cofactor system in carbon dioxide gas‐liquid flow

Formate dehydrogenase from Candida boidinii (CbFDH) is potentially applicable in reduction of CO₂ through oxidation of cofactor NADH into NAD⁺. For this, the CbFDH activity needs to be maintained under practical reaction conditions, such as CO₂ gas‐liquid flow. In this work, CbFDH and cofactor were encapsulated in liposomes and the liposomal enzymes were characterized in an external loop airlift bubble column. The airlift was operated at 45°C with N₂ or CO₂ as gas phase at the superficial gas velocity U_G of 2.0 or 3.0 cm/s. The activities of liposomal CbFDH/cofactor systems were highly stable in the airlift regardless of the type of gas phase because liposome membranes prevented interactions of the encapsulated enzyme and cofactor molecules with the gas‐liquid interface of bubbles. On the other hand, free CbFDH was deactivated in the airlift especially at high U_G with CO₂ bubbles. The liposomal CbFDH/NADH could catalyze reduction of CO₂ in the airlift giving the fractional oxidation of the liposomal NADH of 23% at the reaction time of 360 min. The cofactor was kept inside liposomes during the reaction operation with less than 10% of leakage. All of the results obtained demonstrate that the liposomal CbFDH/NADH functions as a stable catalyst for reduction of CO₂ in the airlift. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Genetic analysis of nitrate reductase deficient mutants of Nicotiana plumbaginifolia: Evidence for six complementation groups among 70 classified molybdenum cofactor deficient mutants

Summary A total of 70 cnx mutants have been characterized from a collection of 211 nitrate reductase deficient (NR^-) mutants isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures after chlorate selection and regeneration into plants. They are presumed to be affected in the biosynthesis of the molybdenum cofactor since they are also deficient for xanthine dehydrogenase activity but contain NR apoenzyme. The remaining clones were classified as nia mutants. Sexual crosses performed between cnx mutants allowed them to be classified into six independent complementation groups. Mutants representative of these complementation groups were used for somatic hybridization experiments with the already characterized N. plumbaginifolia mutants NX1, NX24, NX23 and CNX103 belonging to the complementation groups cnxA, B, C and D respectively. On the basis of genetic analysis and somatic hybridization experiments, two new complementation groups, cnxE and F, not previously described in higher plants, were characterized. Unphysiologically high levels of molybdate can restore the NR activity of cnxA mutant seedlings in vivo, but cannot restore NR activity to any mutant from the other cnx complementation groups.     

Crop residue application increases nitrogen fixation and dry matter production in groundnut (Arachis hypogaea L.) grown on an acid sandy soil in Niger,West Africa

Field experiments were conducted during the rainy reasons of 1989, 1990 and 1991 on an acid sandy soil in Niger, West Africa, to assess the effect of millet straw application (+CR) on growth and N₂ fixation of groundnut (Arachis hypogaea L.).Three years of +CR (4 t ha^–1 yr^–1) increased symbiotic N₂ fixation, total dry matter production (haulm plus pods) by 83% and total nitrogen (N) accumulation by 100%. Concentration of N in the shoot dry matter and total N in the soil were only slightly affected by the +CR treatment.Crop residue application increased the concentration of potassium (K) and molybdenum (Mo) and decreased the concentrations of aluminium (Al) and manganese (Mn) distinctly, both in the plant (shoot and nodule dry matter) and in the soil.The increase in dry matter production and N uptake was mainly due to improved N₂ fixation reflected by enhanced formation and growth of nodules as well as nitrogenase activity. This was attributed to improved chemical soil conditions, particularly to the higher availability of Mo and the lowered content of available Al and Mn.Although with the application of 4 t CR ha^–1, 60 kg K were supplied, increased growth could not be attributed to the additional supply of K.ICRISAT Journal Article No. 1229.ICRISAT Journal Article No. 1229.     

Lily cofactor-independent phosphoglycerate mutase: purification, partial sequencing, and immunolocalization

A cofactor-independent phosphoglycerate mutase (PGAM-i) was isolated to homogeneity from monocotyledonous Lilium longiflorum Thunb. Two-dimensional (2D) polyacrylamide gel electrophoresis resolved three PGAM-i forms. This enzyme was originally identified by cross-reactivity to antibodies affinity-purified from 2D gels using human vitronectin (VN). Antibody produced against a denatured protein spot from a 2D gel did not recognize VN protein, but partial protein and DNA sequencing showed similarity of the former protein to maize PGAM-i. Immunoblots from roots, styles, leaves, and anthers showed the presence of PGAM-i in all tissues examined; it was isolated predominantly from the soluble cell fraction, with some present in the insoluble cell fraction. Immunoelectron microscopy demonstrated its localization in the cytoplasm and plastids in root cells near the apical meristem. In addition, immunogold labeling detected signals from the nucleus. The immunohistochemical localization of the enzyme in the nucleus, as well as in the cytosol and plastids, indicates that lily PGAM-i might have multiple functions in the cell.Abbreviations ELISA enzyme-linked immunosorbent assay - PGAM cofactor-dependent phosphoglycerate mutase - PGAM-i cofactor independent phosphoglycerate mutase - TEM transmission electron microscopy - 2D two dimensional - VN vitronectin     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.     

Analysis of Phaseolus–Rhizobium interactions in a subsistence farming system

Poor bean yields in the Cunha region of the Mata Atlântica ecosystem in the state of São Paulo, Brazil, are associated with low agronomic inputs, plant disease, and soil erosion. To identify sustainable farming practices that increase production and maximize biological N2 fixation (BNF), the effects of soil fertility and plant cultivar on seed yield and root nodule formation were measured under standard agronomic practices. Results from 16 sites showed that fertilizing with lime and molybdenum increased seed yields to 370% for the landrace Serro Azul. In addition to increased yields, plants grown with fertilizer had more nodules. Marked strains of Rhizobium tropici were tested under controlled environments. An indicator strain of Rhizobium containing the gusA marker gene was used. Our results verify that the indicator strain CM-255 GusA+Hup+ had a high capacity to associate with the five bean varieties tested. Fertilization with P, K, S + micronutrients and liming were essential for better nodulation by the indicator strain. Under low fertility conditions, the landrace variety Serro Azul was poorly nodulated, when associated with native strains or with the indicator strain. However, under better soil fertility conditions, nodulation of Serro Azul by the marked Rhizobium strain was increased. The commercial variety Carioca 80SH showed no increase in nodulation (nodule number).     

The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase.

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases. Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis. The Y121F mutant retained 36% of the wild-type activity and the Km value for substrate glycine increased 34-fold, while the activity of the Y121H mutant decreased to 5% of the wild-type activity and the Km value for glycine increased fivefold. The pKa1 values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121F, and Y121H, respectively. The UV-visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 microM for Y121F, Y121H, and the wild-type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase.     

Molybdenum reductase in Enterobacter cloacae

Under anaerobic conditions in glucose–yeast extract medium with phosphate, Enterobacter cloacae strain 48 grew well and reduced Mo6+, to Mo5+. The activity of Mo6+-reductase was measured by the formation of molybdenum blue (complexation between Mo5+ and phosphate ion). Models based on logistic and Luedeking–Piret equations were found adequate to describe the growth of E. cloacae and Mo6+-reductase production. Mo6+-reductase production was found to be a growth-associated process. Washed intact cells, membrane fraction (after disruption using a sonicator) and fluid supernatant (after cell disruption) were able to reduce Mo6+. However, Mo6+-reductase activity was much lower in the supernatant fluid. The (NH4)2SO4-precipitated Mo6+-reductase extract from fluid supernatant was assayed for its properties. The optimum pH and temperature for Mo6+-reductase activity were 8 and 30°C, respectively. The apparent Michaelis–Menten constant (Km) and a maximum velocity (Vmax) were 16.5mm and 0.0192mol/ml.h, respectively.     

New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S（CFP）3A or Venus, respectively, and enable the performance of multicolor BiFC （mcBiFC）. We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.