Characterization of Aspergillus niger pectate lyase A

The Aspergillus niger plyA gene encoding pectate lyase A (EC 4.2.99. 3) was cloned from a chromosomal lambda(EMBL4) library using the Aspergillus nidulans pectate lyase encoding gene [Dean, R. A., and Timberlake, W. E. (1989) Plant Cell 1, 275-284] as a probe. The plyA gene was overexpressed using a promoter fusion with the A. niger pyruvate kinase promoter. Purification of the recombinant pectate lyase A resulted in the identification of two enzyme forms of which one appeared to be N-glycosylated and the other appeared to be free of N-glycosylation. The two enzyme forms showed identical specific activities. The N-glycosylation free pectate lyase A was further characterized with respect to product formation on polygalacturonic acid (alpha-1,4 linked D-galacturonic acid) and mode of action on oligogalacturonides of degree of polymerization 2-8. The bond cleavage frequencies for tetra-, penta-, and hexagalacturonides were studied as a function of [CaCl(2)]. The bond cleavage frequencies changed in a [CaCl(2)]-dependent way for penta- and hexagalacturonide. Kinetic studies using tetra- and hexagalacturonide revealed a strong sigmoidal [CaCl(2)]-dependent relation. The role of Ca(2+) ions in substrate binding is discussed.     

Structure of a plant cell wall fragment complexed to pectate lyase C.

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the beta elimination cleavage of polygalacturonic acid.     

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins

The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.     

Ca2+ binding to alpha-synuclein regulates ligand binding and oligomerization

alpha-Synuclein is a protein normally involved in presynaptic vesicle homeostasis. It participates in the development of Parkinson's disease, in which the nerve cell lesions, Lewy bodies, accumulate alpha-synuclein filaments. The synaptic neurotransmitter release is primarily dependent on Ca(2+)-regulated processes. A microdialysis technique was applied showing that alpha-synuclein binds Ca(2+) with an IC(50) of about 2-300 microm and in a reaction uninhibited by a 50-fold excess of Mg(2+). The Ca(2+)-binding site consists of a novel C-terminally localized acidic 32-amino acid domain also present in the homologue beta-synuclein, as shown by Ca(2+) binding to truncated recombinant and synthetic alpha-synuclein peptides. Ca(2+) binding affects the functional properties of alpha-synuclein. First, the ligand binding of (125)I-labeled bovine microtubule-associated protein 1A is stimulated by Ca(2+) ions in the 1-500 microm range and is dependent on an intact Ca(2+) binding site in alpha-synuclein. Second, the Ca(2+) binding stimulates the proportion of (125)I-alpha-synuclein-containing oligomers. This suggests that Ca(2+) ions may both participate in normal alpha-synuclein functions in the nerve terminal and exercise pathological effects involved in the formation of Lewy bodies.     

Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.     

Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.

The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

A laser Raman spectroscopic study of Ca2+ binding to troponin C.

Laser Raman spectroscopy has been used detect structural changes in troponin C induced by Ca2+ binding. Addition of Ca2+ - Mg2+ sites produces perturbations in the amide III region of the spectrum indicative of increased alpha-helical content, and in regions of the spectrum corresponding to carboxylate, thiol, and phenol side chains. However, Ca2+ binding to the low affinity Ca2+ - specific sites is not detected by laser Raman spectral changes.     

Characterization of a monoclonal antibody against active pectate lyase from Erwinia carotovora

A monoclonal antibody (2E2) produced against pectate lyase from Erwinia carotovora ssp. carotovora reacted with a 41- and a 44-kilodaltion protein on Western blots of concentrated Erwinia culture supernatants resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. It was unequivocally shown that monoclonal 2E2 reacted with an active form of pectate lyase by affinity purifying the antigen with the monoclonal. The affinity-purified antigen was enzymatically active and moved as a single protein band in a nonequilibrium isoelectric focusing gel. Monoclonal 2E2 reacted with the pectate lyases of a diverse range of E. carotovora ssp. carotovora, ssp. atroseptica, and ssp. betavasculorum strains, as well as with one of three strains of E. chrysanthemi. The electrophoretic mobility of the major protein (44 kilodaltons) that reacted with 2E2 was identical within a subspecies but differed among subspecies.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin 1

Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.     

Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-CoA carboxylase by Ca2+ and calmodulin-dependent multiprotein kinase and Ca2+ and phospholipid-dependent protein kinase

ATP-citrate lyase and acetyl-CoA carboxylase purified from lactating rat mammary gland are phosphorylated stoichiometrically by the calmodulin-dependent multiprotein kinase from rabbit skeletal muscle. The reactions are completely dependent on the presence of both Ca2+ and calmodulin. ATP-citrate lyase and acetyl-CoA carboxylase are also phosphorylated stoichiometrically by the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) purified from bovine brain. Phosphorylation of these substrates is stimulated 6-fold and 40-fold respectively by Ca2+ and phosphatidylserine. The calmodulin-dependent and phospholipid-dependent protein kinases phosphorylate the same serine residue on ATP-citrate lyase that is phosphorylated by cyclic-AMP-dependent protein kinase. The sequence of the tryptic peptide containing this site on the mammary enzyme is identical with the sequence of the peptide containing the site on ATP-citrate lyase that is phosphorylated in isolated hepatocytes in response to insulin and/or glucagon. The calmodulin-dependent, phospholipid-dependent and cyclic-AMP-dependent protein kinases phosphorylate distinct sites on acetyl-CoA carboxylase. However, one of the three phosphorylated tryptic peptides derived from enzyme treated with the phospholipid-dependent kinase is identical with the major phosphopeptide (T1) derived from enzyme treated with cyclic-AMP-dependent protein kinase. Phosphorylation of acetyl-CoA carboxylase by the phospholipid-dependent protein kinase inactivates acetyl-CoA carboxylase in a similar manner to cyclic-AMP-dependent protein kinase. With either protein kinase slightly greater phosphorylation and inactivation is seen after pretreatment of acetyl-CoA carboxylase with protein phosphatase-2A, but the effects of the protein phosphatase treatment are not completely reversed. Inactivation by the phospholipid-dependent protein kinase is Ca2+- and phospholipid-dependent, is reversed by protein phosphatase-2A, and correlates with the degree of phosphorylation. The relevance of these findings to insulin- and growth-factor-promoted phosphorylation of ATP-citrate lyase and acetyl-CoA carboxylase in intact cells is discussed.     

Ca2+ and ionic strength dependencies of S1-ADP binding to actin-tropomyosin-troponin: regulatory implications

Skeletal and cardiac muscle contraction are inhibited by the actin-associated complex of tropomyosin-troponin. Binding of Ca(2+) to troponin or binding of ATP-free myosin to actin reverses this inhibition. Ca(2+) and ATP-free myosin stabilize different tropomyosin-actin structural arrangements. The position of tropomyosin on actin affects the binding of ATP-free myosin to actin but does not greatly affect myosin-ATP binding. Ca(2+) and ATP-free myosin alter both the affinity of ATP-free myosin for actin and the kinetics of that binding. A parallel pathway model of regulation simulated the effects of Ca(2+) and ATP-free myosin binding on both equilibrium binding of myosin-nucleotide complexes to actin and the general features of ATPase activity. That model was recently shown to simulate the kinetics of myosin-S1 binding but the analysis was limited to a single condition because of the limited data available. We have now measured equilibrium binding and binding kinetics of myosin-S1-ADP to actin at a series of ionic strengths and free Ca(2+) concentrations. The parallel pathway model of regulation is consistent with those data. In that model the interaction between adjacent regulatory complexes fully saturated with Ca(2+) was destabilized and the inactive state of actin was stabilized at high ionic strength. These changes explain the previously observed change in binding kinetics with increasing ionic strength.     

Characterization and high-level expression of a metagenome-derived alkaline pectate lyase in recombinant Escherichia coli

Alkaline pectate lyases (PLs) play an important role in mild and eco-friendly bioscouring pretreatment processes in the textile industry. However, to date, only a few PLs can be applied in industrial-scale production, and many of them exhibit high production cost, low activity, and/or do not meet the treatment requirements. In this study, an alkaline PL gene was cloned from the metagenomic DNA of alkaline environment soils. The gene pelB consisted of 1263 nucleotides and encoded a mature protein (PelB) of 399 amino acids, which was expressed in Escherichia coli. The maximum catalytic activity of the enzyme exhibited a bimodal distribution at pH 8.1 and 9.8 and an optimal temperature of 55 °C. The K_m and V_max values of PelB were 1.78 g/L and 1084.8 μmol/(L min) at 45 °C, respectively. Substrate specificity analysis demonstrated the high cleavage capability of PelB on a broad range of substrates of natural methylated pectin. Based on the degradation products, PelB was considered to be an endo-acting lyase. Using high-cell-density cultivation in 7-L bioreactor, the highest PL activity (1816.2 U/mL) was achieved. Thus, the recombinant PelB, with promising properties for use in bioscouring in the textile pretreatment process, should be a potential enzyme for industrial applications.     

Calcium binding to troponin C. II. A Ca2+ ion titration study with a Ca2+ ion sensitive electrode

The effects of pH,Mg2+, and ionic strength on Ca2+ binding to rabbit skeletal troponin C were studied by using a Ca2+ sensitive electrode. Troponin C has two high affinity and two low affinity sites and the Ca2+ affinity of both sites was increased by increasing pH in a pH range from pH 5.6 to 10.4. The affinity was decreased by increasing ionic strength. The change of the Ca2+ affinity can be explained by the electrostatic interaction between Ca2+ and the protein. At alkaline pH, the four Ca2+ binding sites bind Ca2+ with the same affinity and the distinction between the high and the low affinity sites vanished. This result shows that the difference of the Ca2+ affinity is owing to differences of the secondary or the tertiary structure of the Ca2+ binding sites, not owing to a difference of the primary structures of the Ca2+ binding sites. The two high affinity sites bound two Ca2+ ions cooperatively in neutral pH. The cooperativity was diminished at both acidic and alkaline pH. Mg2+ ion decreased the affinity of the low affinity sites.     

Circular dichroism of the parallel β helical proteins pectate lyase C and E

The pectate lyases, PelC and PelE, have an unusual folding motif, known as a parallel β-helix, in which the polypeptide chain is coiled into a larger helix composed of three parallel β-sheets connected by loops having variable lengths and conformations. Since the regular secondary structure consists almost entirely of parallel β-sheets these proteins provide a unique opportunity to study the effect of parallel β-helical structure on circular dichroism (CD). We report here the CD spectra of PelC and PelE in the presence and absence of Ca²⁺, derive the parallel β-helical components of the spectra, and compare these results with previous CD studies of parallel β-sheet structure. The shape and intensity of the parallel β-sheet spectrum is distinctive and may be useful in identifying other proteins that contain the parallel β-helical folding motif. © 1995 Wiley-Liss, Inc.