Subunit Molecular Weight Research Articles

Gel-expanded to gel-condensed transition in neurofilament networks revealed by direct force measurements

Neurofilaments (NF)--the principal cytoskeletal constituent of myelinated axons in vertebrates--consist of three molecular-weight subunit proteins NF-L (low), NF-M (medium) and NF-H (high), assembled to form mature filaments with protruding unstructured C-terminus side arms. Liquid-crystal gel networks of side-arm-mediated neurofilament assemblies have a key role in the mechanical stability of neuronal processes. Disruptions of the neurofilament network, owing to neurofilament over-accumulation or incorrect side-arm interactions, are a hallmark of motor-neuron diseases including amyotrophic lateral sclerosis. Using synchrotron X-ray scattering, we report on a direct measurement of forces in reconstituted neurofilament gels under osmotic pressure (P). With increasing pressure near physiological salt and average phosphorylation conditions, NF-LMH, comprising the three subunits near in vivo composition, or NF-LH gels, undergo for P > P(c) approximately 10 kPa, an abrupt non-reversible gel-expanded to gel-condensed transition. The transition indicates side-arm-mediated attractions between neurofilaments consistent with an electrostatic model of interpenetrating chains. In contrast, NF-LM gels remain in a collapsed state for P < P(c) and transition to the gel-condensed state at P > P(c). These findings, which delineate the distinct roles of NF-M and NF-H in regulating neurofilament interactions, shed light on possible mechanisms for disruptions of optimal mechanical network properties.

Structural and functional studies on Ycf12 (Psb30) and PsbZ-deletion mutants from a thermophilic cyanobacterium

Ycf12 (Psb30) and PsbZ are two low molecular weight subunits of photosystem II (PSII), with one and two trans-membrane helices, respectively. In order to study the functions of these two subunits from a structural point of view, we constructed deletion mutants lacking either Ycf12 or PsbZ from Thermosynechococcus elongatus, and purified, crystallized and analyzed the structure of PSII dimer from the two mutants. Our results showed that Ycf12 is located in the periphery of PSII, close to PsbK, PsbZ and PsbJ, and corresponded to the unassigned helix X1 reported previously, in agreement with the recent structure at 2.9 Å resolution (A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 Å resolution: role of quinones, lipids, channels and chloride, Nat. Struct. Mol. Biol. 16 (2009) 334–342). On the other hand, crystals of PsbZ-deleted PSII showed a remarkably different unit cell constants from those of wild-type PSII, indicating a role of PsbZ in the interactions between PSII dimers within the crystal. This is the first example for a different arrangement of PSII dimers within the cyanobacterial PSII crystals. PSII dimers had a lower oxygen-evolving activity from both mutants than that from the wild type. In consistent with this, the relative content of PSII in the thylakoid membranes was lower in the two mutants than that in the wild type. These results suggested that deletion of both subunits affected the PSII activity, thereby destabilized PSII, leading to a decrease in the PSII content in vivo. While PsbZ was present in PSII purified from the Ycf12-deletion mutant, Ycf12 was present in crude PSII but absent in the finally purified PSII from the PsbZ-deletion mutant, indicating a preferential, stabilizing role of PsbZ for the binding of Ycf12 to PSII. These results were discussed in terms of the PSII crystal structure currently available.

Fractional composition of proteins from seeds of transformed (Trn) Gossypium hirsutum

We studied seeds of Gossypium hirsutum L. (Namangan-77 variety) and G. arboreum L. (D-1331 variety) and seeds of cotton transformed forms (Trn 1 and Trn 2 ) that were produced by micro-injection of sperm cells isolated from sprouted G. arboreum L. pollen tubes into ovaries of G. hirsutumm flowers. Pollen tubes were obtained by sprouting fresh pollen in liquid medium. Sprouted pollen tubes were disintegrated in buffer (pH 7.3) containing saccharose (20%), EDTA (5 mM), and Triton X-100 (0.05%). The whole mixture was filtered through a nylon filter and layered into Percoll [3] or saccharose [4–6] gradients. The layer in which sperm cells collected was determined using a microscope. Changes were made to the literature method [3] for the samples that we used. Thus, nutrient medium was supplemented with Triton X-100 (0.05%), EDTA (5 mM, pH 5.3), and CaCl 2 (0.1 M). This mixture was mixed gently for 30 min. Then, the suspension was filtered through a 20–50 μm filter and rinsed with saccharose solution (20%). Each portion of filtrate (5 mL) was treated with Percoll (500 μL). The whole mixture was layered into a gradient of 15% and 40% Percoll in 20% saccharose. The gradient was centrifuged at 9,000 g and 4°C for 60 min. Starch grains, remnants of pollen grain coatings, cell organelles, and other contaminants were precipitated. Sperm cells that accumulated in the 15% Percoll layer were collected, washed, and concentrated by centrifugation at 3,000 g (4°C, 15 min). The viability of the isolated sperm cells was determined. The resulting material was introduced by microinjection into the ovaries of preliminarily castrated flowers. Seeds from ripe pods were used for electrophoretic analysis of proteins. Seeds were ground to a powder. The powder was defatted with acetone. Proteins were dissolved in PS-buffer and heated for 3 min on a boiling water bath. Bromphenol blue (0.01%) was added. Samples were placed on a gel in a minimum volume (30 μL). Electrophoresis of proteins was carried out at room temperature calculated at 5 mA per tube in a PAAG linear concentration gradient from 10 to 18% [7]. The gel was fixed after the electrophoresis was finished in coumassie P-250 solution (0.1%) in EtOH:AcOH:H 2 O (25:5:70). The excess of dye was removed with acetic acid (7%). Bovine albumin (67,000 Da), ovalbumin (45,000 Da), and cytochrome C (11,700 Da) were used as marker proteins. Molecular weights of subunits were calculated from the relative electrophoretic mobility of standard and studied proteins. Our goal was to compare electrophoresis of total proteins from seeds of parent forms and first and second generation transformed plants (Trn 1 and Trn 2 ). Fertile plants grown from seeds that ripened after microinjection of G. arboretum sperm cells into G. hirsutum flower ovaries had a monotypic morphology for the vegetative and generative organs that was intermediate between the initial parent forms [8]. Electrophoretic analysis showed significant differences among fractions of parent specimens (G. hirsutum and G. arboreum) with respect to the distribution of major fractions (Table 1).

Purification and Mass Spectrometric Characterization of Sesbania aculeata (Dhaincha) Stem Lectin

A glucose specific lectin (STA) was isolated from Sesbania aculeata stem by using Sephadex G-50 affinity column chromatography. The lectin is a glycoprotein having 29 kDa subunit molecular weight. Two dimensional gel electrophoresis analysis revealed that the lectin existed in two isomeric forms with varied carbohydrate content as analyzed by high performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and N-terminal sequence (LDSLSFTYNNFE) analysis of this lectin showed 95% homology with stem lectin SL-I (accession no. AJ585523) from peanut plant. The nucleotide sequence of the lectin (STA) was submitted to the gene bank (accession no. EU263636).

Phaseolin diversity as a possible strategy to improve the nutritional value of common beans (Phaseolus vulgaris)

This article proposes a new way to improve the protein quality of the common bean (Phaseolus vulgaris). It is based on the natural variability found in the different types of phaseolin, its main storage protein (40–50% of the total protein). Despite the fact that it is deficient in methionine content, phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2–6) and molecular weight (40–54 kDa) have been analyzed. The similarity of the amino acid composition among phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in phaseolin susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based on highly-digestible phaseolin types could lead to the production of beans with higher protein quality.

Renal AA amyloidosis secondary to morbid obesity?

Systemic amyloidosis is characterized by extracellular deposits on different organs of insoluble fibrils compounded of low molecular weight subunits coming from a great diversity of serum proteins. Secondary amyloidosis AA is due to fibril deposition composed of fragments of the acute phase reactant serum amyloid A. We report a case of a young patient with morbid obesity and hypertension who was admitted to our hospital for acute renal insufficiency associated with nephrotic range proteinuria which developed while on antibiotic treatment for a respiratory infection. AA Amyloidosis was diagnosed by renal biopsy. Based on recent evidence we hypothesize that morbid obesity could be the underlying cause of the deposit disease.

Diversity of Novel Glutenin Subunits in Bread Wheat (Triticum aestivum L.)

Glutenin is a major determinant of baking performance and viscoelasticity, which are responsible for high-quality bread with a light porous crumb structure of a well-leavened loaf. We analyzed the diversity of glutenin genes from six wheat cultivars (Korean cvs. Keumgang and Jinpum, Chinese cvs. China-108 and Yeonnon-78, and Japanese cvs. Norin-61 and Kantou-107). Glutenins contain two types of isoforms such as high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). Glutenin fractions were extracted from wheat endosperm using Osborne solubility method. A total of 217 protein spots were separated on two-dimensional gel electrophoresis with isoelectric focusing (wide range of pH 3–10). The proteins spots were subjected to tryptic digestion and identified by matrix assisted laser desorption/ionization–time of flight mass spectrometry. HMW-GS (43 isoforms) and LMW-GS (seven isoforms) are directly responsible for producing high-quality bread and noodles. Likewise, all the seed storage proteins are digested to provide nutrients for the embryo during seed germination and seedling growth. We identified the diverse glutenin subunits in wheat cultivars and compared the gluten isoforms among different wheat cultivars according to quality. This work gives an insight on the quality improvement in wheat crop.

Purification and characterization of an extracellular carbonic anhydrase from Pseudomonas fragi

Extracellular carbonic anhydrase was purified from Pseudomonas fragi isolated from CaCO 3 enriched soil samples. The enzyme is induced in presence of CaCO 3 and is envisaged to play an important role in bicarbonate ion transport. The 75% ammonium sulphate dialysate was purified by single step affinity chromatography with 86% yield. It is a trimeric protein having a subunit molecular weight of 31.0 kDa and was stable at pH 7.0–8.5 and temperature 35–45 °C. Lead, mercury and EDTA had an inhibitory effect on CA activity, whereas zinc, iron and cadmium increased it. The presence of esterase activity along with IC 50 of sulphonamides and anionic inhibitors indicated that CA from P. fragi belonged to α-class. The CA stability in presence of different salts, as well as in alkaline pH and high temperature makes it a potential candidate to be exploited for biomimetic CO 2 sequestration.

Crystal structure and association behaviour of the GluR2 amino-terminal domain

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.

Application of Capillary Electrophoresis to Determine the Technological Properties of Wheat Flours by a Glutenin Index

Capillary electrophoresis was used to characterize glutenin proteins from ancient varieties of Southern Italy common wheat and to determine the technological properties of wheat flours based on a glutenin index. Three zones were identified in the electropherograms, indicated as A, B, and C according to electroelution order. The three zones corresponded to the low molecular weight glutenin subunits and to the y- and x-type high molecular weight subunits, respectively. The ratio B/C was correlated to the alveographic parameter P/L. These results indicated that flours resulting in a B/C ratio lower than 2 produced elastic doughs whereas flours resulting in a B/C ratio higher than 2 produced doughs more resistant to extension. This study showed that capillary electrophoresis is useful for determining the types and quantities of gluten proteins in the evaluation of wheat-flour technological properties of a limited number of noncommercial varieties as evidenced by the x-type content which seems to strongly influence the flour technological parameters.

A lectin from Sesbania aculeata (Dhaincha) roots and its possible function

A lectin was isolated from the roots of Sesbania aculeata. This is a glucose specific lectin having 39 kDa subunit molecular weight. The expression of this lectin was found to be developmentally regulated and observed to be the highest in the second week. The lectin was purified by affinity chromatography using Sephadex G-50 and found to have 28% homology with Arabidopsis thaliana lectin-like protein (accession No. CAA62665). The lectin binds with lipopolysaccharide isolated from different rhizobial strains indicating the plants interaction with multiple rhizobial species.

A FRET-based Assay To Measure Molecular Distances Within The Ryanodine Receptor Type 1 (RyR1)

The type 1 ryanodine receptor (RyR1) is an intracellular Ca2+-release channel that mediates excitation contraction coupling in skeletal muscle. This enormous homotetrameric protein has a subunit molecular weight of 565 kDa and is associated with numerous regulatory proteins that modulate its function in vivo. Understanding the structure and conformational dynamics of this immense macromolecular complex is a key topic in skeletal muscle biology. In this report, a novel structural assay has been devised that relies upon Forster resonance energy transfer (FRET) to measure distances between defined primary sequence elements of RyR1. These FRET measurements require site-specific incorporation of a fluorescence donor and a fluorescence acceptor into the primary sequence of RyR1. In this system, green fluorescent protein (GFP) fused to the N-terminus of RyR1 acts as the fluorescence donor. The FRET acceptor is an affinity reagent that site-specifically binds to poly-histidine segments (i.e. His tags) engineered into RyR1 where it can accept fluorescence energy from the N-terminal GFP. This report describes the characterization of the FRET acceptor as well as the recombinant His-tagged GFP-RyR1 fusion proteins used for these measurements. In addition, experiments measuring FRET from the N-terminal GFP to the FRET acceptor targeted to His tags introduced into 3 primary sequence elements poorly conserved among the 3 RyR isoforms (i.e. divergent regions) are described. (Supported by NIH grants K01ARO52120 and R21ARO56406).

Trafficking of storage proteins in developing grain of wheat

The processing properties of the wheat flour are largely determined by the structures and interactions of the grain storage proteins (also called gluten proteins) which form a continuous visco-elastic network in dough. Wheat gluten proteins are classically divided into two groups, the monomeric gliadins and the polymeric glutenins, with the latter being further classified into low molecular weight (LMW) and high molecular weight (HMW) subunits. The synthesis, folding and deposition of the gluten proteins take place within the endomembrane system of the plant cell. However, determination of the precise routes of trafficking and deposition of individual gluten proteins in developing wheat grain has been limited in the past by the difficulty of developing monospecific antibodies. To overcome this limitation, a single gluten protein (a LMW subunit) was expressed in transgenic wheat with a C-terminal epitope tag, allowing the protein to be located in the cells of the developing grain using highly specific antibodies. This approach was also combined with the use of wider specificity antibodies to compare the trafficking and deposition of different gluten protein groups within the same endosperm cells. These studies are in agreement with previous suggestions that two trafficking pathways occur in wheat, with the proteins either being transported via the Golgi apparatus into the vacuole or accumulating directly within the lumen of the ER. They also suggest that the same individual protein could be trafficked by either pathway, possibly depending on the stage of development, and that segregation of gluten proteins both between and within protein bodies may occur.

Purification and Characterization of Glutathione-S-Transferase from Chicken Erythrocyte

The glutathione-s-transferases are a family of multifunctional enzymes involved in the detoxification of electrophilic xenobiotics primarily through conjugation to reduce glutathione. A form of the enzyme, designated GSH-S transferase rho, was purified chicken erythrocyte by acetone precipitation, ethanol-chloroform treatment, DEAE-Cellulose, Q-Sepharose, Sephadex G-100 chromatography. The molecular weight of GST purified from chicken erythrocyte was estimated as 47,500 Da by gel filtration. The subunit molecular weight of chicken erythrocyte GST as determined by electrophoresis in the presence of sodium dodecyl sulfate was predicted as 24,000 Da. The specific activity was found to be 20.39 U/mg. The km for CDNB calculated from Lineweaver-Burk plot was 0.71 mM. Optimum temperature of maximum GST activity was 28 degrees C for CDNB. The maximal activity of the enzyme was observed at pH 7.5. The activity of purified GST is inhibited by DDT, urea, CDNB, Triton X-100, DTNB.

Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

In cynobacteria and higher plants, salinity is known to inhibit the activity of several enzymes involved in photosynthesis and hence decreases the overall photosynthetic rate. This gave us an impetus to search for a protease, which may be involved in the turnover of non-functional enzymes produced under salinity stress. Taking the possible changes in pH gradient of the chloroplast under consideration, we have tried to identify a protease, which is induced under salinity and characterized it as an alkaline protease using spinach (Spinacia oleracea) leaves as a model system. The HIC-HPLC purified homogeneous alkaline serine protease from the isolated spinach chloroplasts had two subunits of molecular weight 63 and 32 kDa. The enzyme was maximally active at pH 8.5 and 50°C. The enzyme showed the property to hydrolyze the synthetic substrate like azocaesin and had sufficient proteolytic activity in gelatin bound native PAGE. The enzyme activity was also dependent upon the presence of divalent cations and reduced environment. The active site residues were identified and the homogeneous alkaline serine protease had cysteine, lysine and tryptophan residues at its active site.

Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae)

The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 µg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL 1 and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs.

Detailed Analysis of the Expression of an Alpha-gliadin Promoter and the Deposition of Alpha-gliadin Protein During Wheat Grain Development

Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (coeliac) disease (CD). The B-genome-encoded alpha-gliadin genes, however, contain very few epitopes. Controlling alpha-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of alpha-gliadin protein during wheat grain development. A 592-bp fragment of the promotor of a B-genome-encoded alpha-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an alpha-gliadin-specific antibody was used to detect alpha-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available alpha-gliadin promoter sequences. GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The alpha-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An alpha-gliadin-specific antibody detected alpha-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no alpha-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of alpha-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream. The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the alpha-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how alpha-gliadin expression can be controlled.

Aromatizing Cyclohexa-1,5-diene-1-carbonyl-Coenzyme A Oxidase: CHARACTERIZATION AND ITS ROLE IN ANAEROBIC AROMATIC METABOLISM

Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive de-aromatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M(r) 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K(m) = 10 microm) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H(2)O. DCO was highly specific for 1,5-dienoyl-CoA (K(m) = 27 microm). The initial rate of DCO followed a Nernst curve with half-maximal activity at +10 mV. We propose that DCO provides protection for the extremely oxygen-sensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile de-aromatization/re-aromatization reactions catalyzed by BCR and DCO.

Purification and Some Kinetic Properties of Carbonic Anhydrase from Rainbow Trout (Oncorhynchus mykiss) Liver and Metal Inhibition

In the present study, carbonic anhydrase (CA) enzyme was purified from rainbow trout (RT) liver with a specific activity of 4318 EUxmg(-1) and a yield of 38% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 2260-fold. To check the purity and determine subunit molecular weight of enzyme, SDS-polyacrylamide gel electrophoresis was performed, which showed a single band and MW of approx. 29.4 kDa. The molecular weight of native enzyme was estimated to be approx. 31 kDa by Sephadex-G 200 gel filtration chromatography. Optimum and stable pH were determined as 9.0 in 1 M Tris-SO(4) buffer and 8.5 in 1 M Tris-SO(4) buffer at 4 degrees C, respectively. The optimum temperature, activation energy (E(a)), activation enthalpy ((DeltaH) and Q(10) from Arrhenius plot for the RT liver CA were 40 degrees C, 2.88 kcal/mol, 2.288 kcal/mol and 1.53, respectively. The purified enzyme had an apparent K(m) and V(max) of 0.66 mM and 0.126 micromol x min(-1) for 4-nitrophenylacetate, respectively. K(cat) of the CA was found to be 32.8 s(-1). The inhibitory effects of low concentrations of different metals (Co(II), Cu(II), Zn(II) and Ag(I)) on CA activity were determined using the esterase method under in vitro conditions. The obtained IC(50) values, 50% inhibition of in vitro enzyme activity, were 0.03 mM for cobalt, 30 mM for copper, 47.1 mM for zinc and 0.01 mM for silver. K(i) values for these substances were also calculated from Linewaever-Burk plots as 0.050 mM for cobalt, 1.950 mM for copper, 7.035 mM for zinc and 2.190 mM for silver respectively and determined that cobalt and zinc inhibit the enzyme a competitive manner and copper and silver inhibit the enzyme in an uncompetitive manner.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase.

BackgroundThe emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product.ResultsIn the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting.ConclusionThis is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents.