Molecular characterization of cycloinulooligosaccharide fructanotransferase from Bacillus macerans

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of beta-(2-->1)-linked D-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from Bacillus macerans CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated Mr of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B. macerans CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from B. macerans CFC1. The CFT107 enzyme was produced by B. macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their Km values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (kcat/Km) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.     

Characterization of cyclofructans from inulin by Saccharomyces cerevisiae Strain displaying cell-surface cycloinulooligosaccharide fructanotransferase

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans (GenBank access code AF222787) was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane-anchored protein Aga1p. The surface display of CFTase was confirmed by immunofluorescence microscopy and enzymatic assay. The optimized reaction conditions of surface-displayed CFTase were as follows; pH, 8.0; temperature, 50 degrees C; enzyme amount, 30 milliunit; substrate concentration, 5%; inulin source, Jerusalem artichoke. As a result of the reaction with inulin, cycloinulohexaose was produced as a major product along with cycloinuloheptaose and cycloinulooctaose as minor products.     

类芽孢杆菌环果寡糖糖基转移酶的分离纯化和酶学性质

采用硫铵沉淀、疏水层析和DEAE离子交换层析对产自类芽孢杆菌Paenibacillus sp.Lfos16的环果寡糖糖基转移酶(Cycloinulooligosaccharide Fructanotransferase,CFTase)进行分离纯化,得到电泳纯的CFTase,最终纯化倍数为13. 9,比活力为33. 3 U/mg。纯化的CFTase经SDSPAGE和Native-PAGE电泳分析得出其相对分子量大小约为120 000,且是双亚基蛋白。探究了CFTase的酶学性质,最适反应条件为:40℃～45℃,p H 7. 0;温度稳定范围为30℃～45℃,p H稳定范围为5. 0～9. 0。并对CFTase降解菊糖的催化机制进行了分析,初步确定为外切加环化作用形成环果寡糖。     

The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc-dependent binding to an anionic surface.

The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 microM and 27 microM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M(r) was 7.3 kDa (range 6.2-8.1 kDa). Based on N-terminal sequencing, this polypeptide (1(2)), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 microM. The synthetic peptide HGLGHGH representing the N-terminal of the 1(2)) fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459-475) included within the 1(2) fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 microM in the absence and presence of added Zn2+ (30 microM). The specific binding of 125I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 microM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 microM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the 1(2) fragment was found to inhibit 50% of the 125I-HK binding to kaolin at a concentration of 380 microM. These results suggest that residues contained within the 1(2) fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.     

A synthetic peptide of the N-terminus of actin interacts with myosin

Research reported from numerous laboratories suggested that the N-terminal region of actin contained one of the binding sites between actin and myosin. A synthetic peptide corresponding to residues 1-28 of skeletal actin was prepared by solid-phase peptide methodology. The formation of a complex between this peptide and myosin subfragment 1 (S1) was demonstrated by high-performance size-exclusion chromatography (pH 6.8). The actin peptide precipitated S1 at higher pH (7.4-8.2) but remained soluble when bound to heavy meromyosin (HMM) or S1 in the presence of F-actin. The actin peptide 1-28 bound to S1 and HMM and activated the ATPase activity in a manner similar to that of F-actin. These results demonstrate that the N-terminal region of actin, residues 1-28, contains a biologically important binding site for myosin.     

Heparin-binding histidine and lysine residues of rat selenoprotein P

Selenoprotein P is a plasma protein that has oxidant defense properties. It binds to heparin at pH 7.0, but most of it becomes unbound as the pH is raised to 8.5. This unusual heparin binding behavior was investigated by chemical modification of the basic amino acids of the protein. Diethylpyrocarbonate (DEPC) treatment of the protein abolished its binding to heparin. DEPC and [(14)C]DEPC modification, coupled with amino acid sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry of peptides, identified several peptides in which histidine and lysine residues had been modified by DEPC. Two peptides from one region (residues 80-95) were identified by both methods. Moreover, the two peptides that constituted this sequence bound to heparin. Finally, when DEPC modification of the protein was carried out in the presence of heparin, these two peptides did not become modified by DEPC. Based on these results, the heparin-binding region of the protein sequence was identified as KHAHLKKQVSDHIAVY. Two other peptides (residues 178-189 and 194-234) that contain histidine-rich sequences met some but not all of the criteria of heparin-binding sites, and it is possible that they and the histidine-rich sequence between them bind to heparin under some conditions. The present results indicate that histidine is a constituent of the heparin-binding site of selenoprotein P. The presence of histidine, the pK(a) of which is 7.0, explains the release of selenoprotein P from heparin binding as pH rises above 7.0. It can be speculated that this property would lead to increased binding of selenoprotein P in tissue regions that have low pH.     

Purification and characterization of a heat stable inulin fructotransferase (DFA I-producing) from Arthrobacter pascens a62-1

An inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter pascens a62-1 was purified and the properties of the enzyme were investigated. The enzyme was purified from culture supernatant of the microorganism 58.5 fold with a yield of 8.32% using Super Q Toyopearl chromatography and butyl Toyopearl chromatography. It showed maximum activity at pH 5.5 and 45 °C and was stable up to 75 °C. This heat stability was highest in the inulin fructotransferases (DFA I-producing) reported until now. The molecular mass of the enzyme was estimated to be 37,000 by SDS-PAGE and 60,000 by gel filtration, and was considered to be a dimer. The N-terminal amino acid sequence (20 amino acid residues) was determined as Ala-Asn-Thr-Val-Tyr-Asp-Val-Thr-Thr-Trp-Ser-Gly-Ala-Thr-Ile-Ser-Pro-Tyr-Val-Asp.     

Recombinant thermostable cycloinulo-oligosaccharide fructanotransferase produced by Saccharomyces cerevisiae.

A truncated fragment of the cycloinulo-oligosaccharide fructanotransferase (CFTase) gene of Bacillus circulans MCI-2554 was fused to the prepro secretion sequence of the alpha-factor and expressed in Saccharomyces cerevisiae under the control of the 5' upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL). Efficiently secreted recombinant CFTase protein (yeast CFTase) was purified. Yeast CFTase consisted of three protein molecules, each of which had CFTase activity (yeast CFTase 1 [116 kDa], yeast CFTase 2 [117 kDa], and yeast CFTase 3 [116 kDa]). Yeast CFTase 2 was the major product of the expression system employed and was shown to be N glycosylated by endoglycosidase H treatment. Yeast CFTase 1 was N glycosylated but had a short truncation at its N terminus, while yeast CFTase 3 did not contain an N-glycosylated carbohydrate chain(s). Yeast CFTase 2 showed an optimum pH, an optimum temperature, and a pH stability similar to those of CFTase purified from B. circulans but exhibited a significant increase in thermostability. Production of yeast CFTase by the strain which had two copies of the CFTase gene integrated into its chromosomes reached 391 U per liter of culture at 120 h, which corresponded to 8.40 mg of protein per liter, by shake-flask cultivation.     

Metal binding and metal-induced conformational change of frog bone gamma-carboxyglutamic acid-containing protein

Ca2+ or Cd2+ binding and the conformational change induced by the metal binding in two frog bone Gla-proteins (BGP, termed BGP-1 and BGP-2) were studied by equilibrium dialysis and CD measurement. By CD measurement in the far-ultraviolet region, the alpha-helix content of both apoBGPs was found to be 8%. Binding of both Ca2+ and Cd2+ was accompanied with a change in the CD spectrum, and the alpha-helix content increased to 15 and 25% for BGP-1 and BGP-2, respectively. CD measurement in the near-ultraviolet region indicated that the environment of aromatic amino acid residues in the protein molecule was changed by metal binding. Equilibrium dialysis experiments indicated that each of these two protein binds specifically 2 mol of Ca2+, and nonspecifically an additional 3-4 mol of Ca2+ in 0.02 M Tris-HCl/0.15 M NaCl (pH 7.4), at 4 degrees C. According to the two separate binding sites model, BGP-1 has 1 high-affinity Ca2+ binding site (Kd1 = 0.17 mM) and 1 low-affinity site (Kd2 = 0.29 mM), and BGP-2 contains 1 high-affinity site (Kd1 = 0.14 mM) and 1 low-affinity site (Kd2 = 0.67 mM). In addition, 2 Cd2+ bound to a high-affinity binding site on BGP-1 with Kd1 of 10.4 microM, and 1 Cd2+ bound to a low-affinity binding site with Kd2 of 41.5 microM. On the other hand, BGP-2 had three classes of binding sites and 1 Cd2+ bound to each binding site with Kd1 = 3.6 microM, Kd2 = 16.3 microM, Kd3 = 51.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a membrane-bound aminopeptidase purified from Acyrthosiphon pisum midgut cells. A major binding site for toxic mannose lectins

A single membrane-bound aminopeptidase N (APN) occurs in the pea aphid (Acyrthosiphon pisum Harris) midgut, with a pH optimum of 7.0, pI of 8.1 and molecular mass of 130 kDa. This enzyme accounts for more than 15.6% of the total gut proteins. After being solubilized in detergent, APN was purified to homogeneity. The enzyme is a glycoprotein rich in mannose residues, which binds the entomotoxic lectins of the concanavalin family. The internal sequence of APN is homologous with a conservative domain in APNs, and degenerated primers of highly conserved APN motifs were used to screen a gut cDNA library. The complete sequence of APN has standard residues involved in zinc co-ordination and catalysis and a glycosyl-phosphatidylinositol anchor, as in APNs from Lepidoptera. APN has a broad specificity towards N-terminal amino acids, but does not hydrolyze acidic aminoacyl-peptides, thus resembling the mammalian enzyme (EC 3.4.11.2). The kcat/Km ratios for different di-, tri-, tetra-, and penta-peptides suggest a preference for tripeptides, and that subsites S1, S2' and S3' are pockets able to bind bulky aminoacyl residues. Bestatin and amastatin bound APN in a rapidly reversible mode, with Ki values of 1.8 microM and 0.6 microM, respectively. EDTA inactivates this APN (k(obs) 0.14 M(-1) x s(-1), reaction order of 0.44) at a rate that is reduced by competitive inhibitors. In addition to oligopeptide digestion, APN is proposed to be associated with amino-acid-absorption processes which, in contrast with aminopeptidase activity, may be hampered on lectin binding.     

Crystal Structure of Family 14 Polysaccharide Lyase with pH-dependent Modes of Action

The Chlorella virus enzyme vAL-1 (38 kDa), a member of polysaccharide lyase family 14, degrades the Chlorella cell wall by cleaving the glycoside bond of the glucuronate residue (GlcA) through a β-elimination reaction. The enzyme consists of an N-terminal cell wall-attaching domain (11 kDa) and a C-terminal catalytic module (27 kDa). Here, we show the enzyme characteristics of vAL-1, especially its pH-dependent modes of action, and determine the structure of the catalytic module. vAL-1 also exhibited alginate lyase activity at alkaline pH, and truncation of the N-terminal domain increased the lyase activity by 50-fold at pH 7.0. The truncated form vAL-1(S) released di- to hexasaccharides from alginate at pH 7.0, whereas disaccharides were preferentially generated at pH 10.0. This indicates that vAL-1(S) shows two pH-dependent modes of action: endo- and exotypes. The x-ray crystal structure of vAL-1(S) at 1.2 Å resolution showed two antiparallel β-sheets with a deep cleft showing a β-jelly roll fold. The structure of GlcA-bound vAL-1(S) at pH 7.0 and 10.0 was determined: GlcA was found to be bound outside and inside the cleft at pH 7.0 and 10.0, respectively. This suggests that the electric charges at the active site greatly influence the binding mode of substrates and regulate endo/exo activity. Site-directed mutagenesis demonstrated that vAL-1(S) has a specific amino acid arrangement distinct from other alginate lyases crucial for catalysis. This is, to our knowledge, the first study in which the structure of a family 14 polysaccharide lyase with two different modes of action has been determined.     

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative

Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.     

Biochemical characterization of the structure-specific DNA-binding protein Cmb1 from Schizosaccharomyces pombe

Cmb1, a novel HMG box protein from Schizosaccharomyces pombe, has been characterized biochemically using glutaraldehyde cross-linking, gel-filtration and analytical ultracentrifugation. It was identified as a monomeric, non-spherical protein, with a tendency to aggregate in solution. Limited proteolysis with trypsin and chymotrypsin showed that the C-terminal HMG box was a compact, proteolytically stable domain and the N-terminal region of Cmb1 was relatively unstructured and more easily digested.As Cmb1 was previously identified as a potential mismatch-binding protein, the binding constants and stoichiometry for both homoduplex and heteroduplex DNA were determined using an IASys resonant mirror biosensor. Cmb1 indeed demonstrated a tighter association with mismatched DNA, especially with the C/Delta-mismatch. Expression constructs of Cmb1 were made to study the sections of the protein involved in DNA binding. Constructs with the N-terminal region absent revealed that the C-terminal HMG box was the primary DNA-binding region. The presence of the N-terminal region did, however, facilitate tighter binding to both homoduplex and heteroduplex DNA. The amino acid residues isoleucine 14 and leucine 39 were located as putative intercalating residues using structure guided homology modelling. The model templates were derived from two distinct HMG:DNA complexes: HMG-D bound to homoduplex DNA and HMG 1 bound to cisplatin DNA. Binding studies using the Cmb1 HMG box with point mutations in these residues showed that isoleucine 14 was important for the binding of Cmb1 to homoduplex DNA, but affected binding to mismatches to a lesser extent. In contrast, leucine 39 appeared to have a more significant function in binding to mismatched DNA.     

Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits

Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.     

A calcium-43 NMR study of calcium binding to an acidic proline-rich phosphoprotein from human saliva

The 43Ca NMR line width measured for Ca2+ bound to protein A, an acidic proline-rich salivary protein, is 1 order of magnitude narrower than has previously been observed for other proteins of similar molecular weight. The correlation times, quadrupole coupling constants, and chemical shifts estimated for Ca2+ ions bound to the intact protein (Mr approximately 10 000) and its 30 amino acid residue long acidic N-terminal TX peptide were indistinguishable within experimental error. These results--as well as the outcome of 1H NMR relaxation rate measurements--are indicative of extensive motions for the protein residues, which in turn give rise to a high degree of flexibility for the protein-bound Ca2+. Ca2+ titration and pH-dependent measurements on protein A, the TX peptide, and the dephosphorylated TX peptide established the importance of the two phosphoserine residues in the binding of Ca2+. Moreover, a comparison of the 43Ca NMR parameters with those obtained for other Ca2+-binding proteins suggests the presence of Ca2+-binding sites of similar symmetry in all these proteins. No evidence was found for a proposed interaction between the highly acidic N-terminal and the weakly basic C-terminal regions of protein A. In contrast, the high pH inflection that was observed in the pH titration curve for the intact protein was also found for the phospho and dephospho TX peptides, thus suggesting that basic moieties in the N-terminal region rather than those in the C-terminal region may be responsible for this observation.     

Properties of calcium binding by Myxicola axoplasmic protein

The 45Ca2+ binding properties of axoplasmic protein from the Myxicola giant axon have been investigated using a centrifugal/concentration-dialysis technique. Scatchard plot analysis of these binding data suggest that Ca2+ is attached to a site with an equilibrium dissociation constant of 7.7 +/- 0.5 microM and a capacity of 4.4 +/- 0.2 mumol/g axoplasmic protein (n = 11). Addition of other cations--Cd2+, Mn2+, Al3+, Cu2+, Ba2+, and Zn2(+)--at concentrations up to 10 microM did not displace 0.2 microM 45Ca2+ from its binding site, probably because of buffering of these cations by amino acid residues within the protein solutions. The protein could be stored at 4 degrees C for up to 16 days with no appreciable change in the number of calcium sites. Ca2+ binding equilibrium took place in less than 30 min of incubation. Increasing the incubation temperature from 4 degrees C to 37 degree C reduced the number of Ca2+ sites. The binding capacity was reduced by one-half when the protein was dialyzed with 4 M urea or high ionic strength KCl (2 M). Calcium binding was examined as a function of pH. When the protein was dialyzed overnight at different pH values and all the binding was done at pH 7.0, the apparent number of Ca2+ sites decreased as the pH of the dialysis medium was increased. When the protein was dialyzed overnight at pH 7.0 and the binding was done at different pH values, the apparent binding capacity increased as pH increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatitis C virus NS5B RNA replicase specifically binds ribosomes

Hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA replicase. We expressed full-length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP-NS5B). MBP-NS5B was recovered in the soluble fraction after centrifugation at 40,000 x g and affinity-purified with amylose resin. The purified MBP-NS5B had a high-level of poly (A), oligo (U)-dependent UMP incorporation with a Km of 2 microM for UTP. Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160,000 x g. The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP-NS5B. Ribosomes and MBP-NS5B were subsequently co-purified on amylose resin. Deletion study revealed that either the N-terminal (amino acid residues 1-107) or the C-terminal (amino acid residues 498-591) region of NS5B were sufficient for this association with ribosomes. We further found that NS5B also bound with human ribosomes. Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.     

The sensor kinase CitA (DpiB) of Escherichia coli functions as a high-affinity citrate receptor

For the CitA-CitB (DpiB-DpiA) two-component signal transduction system from Escherichia coli, three diverse functions have been reported: induction of the citrate fermentation genes citCDEFXGT, repression of the regulator gene appY, and destabilization of the inheritance of iteron-containing plasmids such as pSC101. This poses the question of the principal biological role of this system. Here it is shown that the periplasmic domain of the E. coli sensor kinase CitA functions as a high-affinity citrate receptor. Two CitA derivatives were purified by affinity chromatography and subjected to binding studies using isothermal titration calorimetry (ITC). One of them, termed CitA215MBP, comprised the N-terminal part of CitA (amino acid residues 1-215), including the two transmembrane helices, and was fused to the amino terminus of the E. coli maltose-binding protein lacking its signal peptide. The second CitA derivative, designated CitAP(Ec), encompassed only the periplasmic domain (amino acid residues 38-177). CitA215MBP bound citrate at 25 degrees C with a K(d) of 0.3 microM and a binding stoichiometry of up to 0.9 in 50 mM sodium phosphate buffer, pH 7. Binding was driven by the enthalpy change (Delta H of -95.7 kJ mol(-1)), whereas the entropy change was not favorable for binding ( T Delta S of -58.6 kJ mol(-1)). ITC experiments with CitAP(Ec) yielded similar K(d) values for citrate (0.15-1.0 microM). Besides citrate, also isocitrate ( K(d) approximately tricarballylate ( K(d) approximately t not malate were bound by CitAP(Ec). The results favor the assumption that the primary biological function of the CitA-CitB system is the regulation of the citrate fermentation genes.     

Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA

A cDNA (1061 bp) Bj glyII was cloned from a mannitol induced library of Brassica juncea. It encoded a protein of 335 amino acids with a molecular weight of 36.52 kDa. The deduced amino acid sequence of the clone showed 92% and 56% identity with Pennisetum and rice glyoxalase II, respectively, and 30% identity was observed with the human glyoxalase II. Search for the identical residues revealed the presence of highly conserved THHHXDH domain which is involved in zinc binding. p-NN and pSORT analysis of this sequence revealed a N-terminal mitochondrial target peptide. The cDNA was cloned in pMAL and a fusion protein with MBP (78 kDa) was expressed in Escherichia coli. The recombinant protein was purified approximately sixfold by affinity purification on amylose column and showed its pH optima at 7.0. The K(m) was determined to be 120 microM using S-d-lactoylglutathione as substrate. The expression of Bj glyII under various abiotic stress conditions showed that it is upregulated by salinity, heavy metal stress, and ABA.     

Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a

The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full-length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA-methylation activities than Dnmt3a2. The N-terminal sequence from residues 1 to 211 was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, Lys51, Lys53, Arg177 and Arg179, in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1-211) was localized in nuclei, whereas that harbouring mutations at the four basic amino acid residues was also detected in the cytoplasm. The DNA-methylation activity of Dnmt3a with the mutations was suppressed under physiological salt conditions, which is similar that of Dnmt3a2. In addition, ectopically expressed Dnmt3a with mutations, as well as Dnmt3a2, could not be retained efficiently in nuclei on salt extraction. We conclude that the DNA-binding activity of the N-terminal domain contributes to the DNA-methyltransferase activity via anchoring of the whole molecule to DNA under physiological salt conditions.