Relative susceptibilities of the glucosamine-glucuronic acid and N-acetylglucosamine-glucuronic acid linkages to heparin lyase III

Heparin lyases are valuable tools for generating oligosaccharide fragments and in sequence determination of heparan sulfate (HS). Heparin lyase III is known to cleave the linkages between N-acetylglucosamine (GlcNAc) or N-sulfated glucosamine (GlcNS) and glucuronic acid (GlcA) as the primary sites and the linkages between GlcNAc, GlcNAc(6S), or GlcNS and iduronic acid as secondary sites. N-Unsubstituted glucosamine (GlcN) occurs as a minor component in HS, and it has been associated with various bioactivities. Here we investigate the specificity of heparin lyase III toward the GlcN-GlcA linkage using a recombinant enzyme of high purity and as substrates the partially de-N-acetylated polysaccharide of Escherichia coli K5 strain and derived hexasaccharides. The specificity of lyase III toward the GlcN-GlcA linkage is deduced by sequencing of the oligosaccharide products using electrospray mass spectrometry with collision-induced dissociation and MS/MS scanning. The results demonstrate that under controlled conditions for partial digestion, lyase III does not act at the GlcN-GlcA linkage, whereas GlcNAc-GlcA is cleaved. Even under forced conditions for exhaustive digestion, the GlcN-GlcA linkage is only partly cleaved. It is this property of lyase III that has enabled the isolation of a unique, nonsulfated antigenic determinant DeltaUA-GlcN-UA-GlcNAc from HS and from partially de-N-acetylated K5 polysaccharide. It was unexpected that pentasaccharide fragments were also detected among the digestion products of the K5 polysaccharide used. It is possible that these are products of an additional glycosidase activity of lyase III, although other mechanisms cannot be completely ruled out.     

Selective cleavage of glycosidic linkages: Studies with the O-specific polysaccharide from Shigella dysenteriae type 3

Treatment of the O-specific polysaccharide from Shigella dysenteriae Type 3 with hydrazine in the presence of hydrazine sulphate resulted in quantitative N-deacetylation with the formation of a modified polysaccharide containing free amino groups. Oxidation of the modified polysaccharide with periodate did not destroy the 2-amino-2-deoxygalactose residues, thus indicating that they were substituted at position 3. Acid hydrolysis of the modified polysaccharide afforded 3-O-(2-amino-2-deoxy-β-D-galactopyranosyl)-D-galactose, which was identified as the N-acetyl derivative. Deamination of the modified polysaccharide with nitrous acid cleaved the 2-amino-2-deoxy-D-galactopyranosyl linkages to give a pentasaccharide as the major product, which appeared to be the modified chemical repeating unit of the O-specific polysaccharide.     

Molecular dynamics study of the conformations of glycosidic linkages in sialic acid modified ganglioside GM3 analogues

The objective of the present study is to model the analogues of monosialoganglioside (GM3) by making modifications in its sialic acid residue with different substitutions in aqueous environment and to determine their structural stability based upon computational molecular dynamics. Molecular mechanics and molecular dynamics investigation was carried out to study the conformational preferences of the analogues of GM3. Dynamic simulations were carried out on the analogues of GM3 varying in the substituents at C-1, C-4, C-5, C-8 and C-9 positions of their sialic acid or Neuraminic acid (NeuAc) residue. The analogues are soaked in a periodic box of TIP3P water as solvent and subjected to a 10 ns molecular dynamics (MD) simulation using AMBER ff03 and gaff force fields with 30 ps equilibration. The analogue of GM3 with 9-N-succNeuAc (analogue5, C9 substitution) was observed to have the lowest energy of ?6112.5 kcal/mol. Graphical analysis made on the MD trajectory reveals the direct and water mediated hydrogen bonds existing in these sialic acid analogues. The preferable conformations for glycosidic linkages of GM3 analogues found in different minimum energy regions in the conformational maps were identified. This study sheds light on the conformational preferences of GM3 analogues which may be essential for the design of GM3 analogues as inhibitors for different ganglioside specific pathogenic proteins such as bacterial toxins, influenza toxins and neuraminidases.     

Methods for the identification of N-asparaginyl and O-seryl/threonyl glycosidic linkages to aminosugars in glycoproteins

Methods are presented for the identification of certain glycopeptide bonds in glycoproteins. Mucin-type linkages are determined following treatment of glycoproteins with alkaline sodium [³H]borohydride. Such treatment cleaves O-glycosidic bonds to serine and threonine and simultaneously labels the sugar and amino acid components of the linkage. Following acid hydrolysis and dansylation, the sugar component of the linkage is identified as its corresponding dansyl-hexosaminitol by fluorographic techniques. A method is described for the separation of dansyl-galactosaminitol and dansyl-glucosaminitol by thin-layer electrophoresis in borate buffers. The amino acid component of the glycopeptide linkage is identified by fluorography following two-dimensional thin-layer chromatography of its dansyl derivative on polyamide plates. For the analysis of plasma-type glycoproteins, glycopeptides are prepared by exhaustive pronase digestion and purified by gel filtration chromatography. Final purification is effected by dansylation and thin-layer electrophoresis. The linkage compound 2-acetamido-1-N-β-l-aspartyl-2-deoxy-β-d-glucopyranosylamine is isolated from such glycopeptides as its dansyl derivative following partial acid hydrolysis. Its identity is confirmed by comparison of its properties with those of the synthetic compound. Thus the components of the glycosylamine linkage are identified following complete acid hydrolysis, redansylation, and separation by thin-layer electrophoresis.     

The rate of spontaneous cleavage of the glycosidic bond of adenosine

Previous estimates of the rate of spontaneous cleavage of the glycosidic bond of adenosine were determined by extrapolating the rates of the acid- and base-catalyzed reactions to neutral pH. Here we show that cleavage also proceeds through a pH-independent mechanism. Rate constants were determined as a function of temperature at pH 7 and a linear Arrhenius plot was constructed. Uncatalyzed cleavage occurs with a rate constant of 3.7 × 10⁻¹² s⁻¹ at 25 °C, and the rate enhancement generated by the corresponding glycoside hydrolase is ∼5 × 10¹²-fold.     

Molecular identification of a polyM-specific alginate lyase from Pseudomonas sp. strain KS-408 for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in alginate

An alginate lyase gene of a newly isolated Pseudomonas sp. strain KS-408 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. A partial protein sequence of KS-408 alginate lyase was homology-modeled on the basis of the crystal structure of A1-III alginate lyase from Sphingomonas sp. strain A1. The proposed 3-D structure of KS-408 alginate lyase shows that Asn-198, His-199, Arg-246, and Tyr-253 residues are conserved for the catalytic active site. The recombinant KS-408-1F (with signal peptide) and KS-408-2F (without signal peptide) alginate lyases with the (His)(6) tag consist of 393 (44.5 kDa) and 372 (42.4 kDa) amino acids with isoelectric points of 8.64 and 8.46, respectively. The purified recombinant KS-408 alginate lyase was very stable when it was incubated at 40 °C for 30 min. Alginate oligosaccharides produced by the KS-408-2F alginate lyase were purified on a Bio-Gel P2 column and analyzed by thin-layer chromatography, fast-protein liquid chromatography, and electrospray ionization mass spectrometry. (1)H NMR data showed that the KS-408-2F alginate lyase cleaved the glycosidic linkages between two mannuronates (mannuronate-β(1-4)-mannuronate) or mannuronate and guluronate (mannuronate-β(1-4)-guluronate), indicating that the KS-408 alginate lyase is a polyM-specific lyase.     

The specific substance from Pneumococcus type 34. The configuration of the glycosidic linkages

1. The specific compound from Pneumococcus type 34 was isolated from capsular material by ion-exchange chromatography. This separated it from a substance with chemical and serological properties corresponding to those reported for C-substance. 2. The configuration of the two galactofuranosyl linkages in the repeating unit of S.34 was determined and the configurations previously assigned to the other glycosidic linkages were confirmed. 3. The dephosphorylated deacetylated repeating unit is thus O-beta-d-galactofuranosyl-(1-->3)-O-alpha-d-glucopyranosyl-(1-->2)-O-beta-d-galactofuranosyl-(1-->3)-O-alpha-d-galactopyranosyl- (1-->2)-ribitol.     

Evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A lyase arginine-41 as a catalytic residue: use of acetyldithio-coenzyme A to monitor product enolization

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to produce acetyl-CoA and acetoacetate. Arginine-41 is an invariant residue in HMG-CoA lyases. Mutation of this residue (R41Q) correlates with human HMG-CoA lyase deficiency. To evaluate the functional importance of arginine-41, R41Q and R41M recombinant mutant human HMG-CoA lyase proteins have been constructed, expressed, and purified. These mutant proteins retain structural integrity based on Mn(2+) binding and affinity labeling stoichiometry. R41Q exhibits a 10(5)-fold decrease in V(max); R41M activity is >or=10-fold lower than the activity of R41Q. Acetyldithio-CoA, an analogue of the reaction product, acetyl-CoA, has been employed to test the function of arginine-41, as well as other residues (e.g., aspartate-42 and histidine-233) implicated in catalysis. Acetyldithio-CoA supports enzyme-catalyzed exchange of the methyl protons of the acetyl group with solvent; exchange is dependent on the presence of Mg(2+) and acetoacetate. In comparison with wild-type human enzyme, D42A and H233A mutant enzymes exhibit 4-fold and 10-fold decreases, respectively, in the proton exchange rate. In contrast, R41Q and R41M mutants do not catalyze any substantial enzyme-dependent proton exchange. These results suggest a role for arginine-41 in deprotonation or enolization of acetyldithio-CoA and implicate this residue in the HMG-CoA cleavage reaction chemistry that leads to acetyl-CoA product formation. Assignment of arginine-41 as an active site residue is also supported by a homology model for HMG-CoA lyase based on the structure of 4-hydroxy-2-ketovalerate aldolase. This model suggests the proximity of arginine-41 to other amino acids (aspartate-42, glutamate-72, histidine-235) implicated as active site residues based on their function as ligands to the activator cation.     

A characterization of the BALB/c lambda and kappa class antibodies to alpha (1,3) glycosidic linkages

The avidity relationships of kappa and lambda antibodies with specificity for the glucosyl-alpha (1,3)-glucose disaccharide epitope were compared using a solid-phase hapten inhibition assay. The kappa class antibodies, induced by the T-cell-dependent antigen, nigerosyl-keyhole limpet hemocyanin (N-KLH), and the lambda class antibodies, induced by the T-cell-independent antigen, Dextran B1355, were only marginally distinguishable (approximately equal to 10-fold) on the basis of hapten inhibition using the free disaccharide, nigerose, as inhibitor. Analysis of the binding site sizes of antibodies induced by N-KLH through inhibition with oligosaccharides of different sizes showed that the disaccharide protein antigen did not select for antibodies with smaller binding site sizes. The cellular basis for these findings is discussed.     

Randomness in the heparin polymer: computer simulations of alternative action patterns of heparin lyase

Heparin is a mixture of linear polysaccharides of undetermined sequence. Both biosynthetic data and computer simulation studies have established that each heparin polymer chain is comprised of oligosaccharides of defined sequence, representing ordered domains. One such ordered domian is a pentasaccharide corresponding to heparin's antithrombin III binding site. Previous computer simulation studies, performed under the assumption that heparin lyase (heparinase, EC 4.2.2.7), has a random endolytic action pattern, suggested that certain of these ordered oligosaccharide domains may themselves be nonrandomly arranged in the heparin polymer. The present work presents computer simulations of alternative action patterns for heparin lyase while assuming a random distribution of these oligosaccharide units within the heparin polymer. We consider action patterns that are determined solely by the primary structure of the substrate molecules. Results of the simulations are compared to (1) the experimental measurements of product chains formed throughout the reaction and (2) the change in weight average molecular weight Mw as a function of reaction completion as determined by absorbance at 232 nm. From the simulation of 60 action patterns for heparin lyase, we infer that one of the following statements concerning heparin and heparin lyase is true: (1) Heparin is a random arrangement of a small number of structurally defined oligosaccharide units. Heparin lyase changes its action pattern during the depolymerization of heparin (perhaps influenced by the secondary structure of substrate). (2) Heparin contain clusters of oligosaccharide sequences that are present in low concentrations (overall) in the polymer. Heparin lyase has a specificity for cleaving glycosidic linkages either exolytically at the nonreducing terminus of a chain or (endolytically) at the reducing side of these rare oligosaccharide sequence.     

Cloning, expression and characterization of acharan sulfate-degrading heparin lyase II from Bacteroides stercoris HJ-15

Aims:  This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15.
Methods and Results:  The heparinase II gene from Bact. stercoris HJ-15 was identified by Southern blotting and the sequence was deposited in GenBank. The gene was cloned and overexpressed in Escherichia coli , and rHepII was purified using two simple ion–exchange column chromatography steps. Enzymatic properties and substrate specificities of rHepII were assessed and its kinetic constants were calculated. Heparin-like glycosaminoglycans (HLGAGs) were digested with rHepII under optimal reaction conditions, and the products were analysed by SAX-HPLC.
Conclusions:  The heparinase II gene is 2322-bp long and consists of 773 amino acids. rHepII is most active in 50 mmol l⁻¹ sodium phosphate buffer with 75 mmol l⁻¹ NaCl (pH 7·4) at 32°C, and the activity is stable at 4°C for 15 days on storage. Acharan sulfate is the best substrate for rHepII, followed by heparan sulfate and heparin. The major degradation products were verified as highly sulfated disaccharides through SAX-HPLC analysis. It means that rHepII prefers iduronic acid over glucuronic acid on the HLGAG structure.
Significance and Impact of the Study:  This study provides easy and certain means for obtaining large amounts of pure rHepII and also provides important information regarding the tendencies of this enzyme and its digested products. rHepII digests HLGAGs in a different manner than heparinases from Flavobacterium heparinum ; therefore, we anticipate that rHepII will be a powerful tool for studies of GAGs and GAGs lyases.     

Phosphorylation of photosystem II core proteins prevents undesirable cleavage of D1 and contributes to the fine‐tuned repair of photosystem II

Photosystem II (PSII) is a primary target for light‐induced damage in photosynthetic protein complexes. To avoid photoinhibition, chloroplasts have evolved a repair cycle with efficient degradation of the PSII reaction center protein, D1, by the proteases FtsH and Deg. Earlier reports have described that phosphorylated D1 is a poor substrate for proteolysis, suggesting a mechanistic role for protein phosphorylation in PSII quality control, but its precise role remains elusive. STN8, a protein kinase, plays a central role in this phosphorylation process. To elucidate the relationship between phosphorylation of D1 and the protease function we assessed in this study the involvement of STN8, using Arabidopsis thaliana mutants lacking FtsH2 [yellow variegated2 (var2)] and Deg5/Deg8 (deg5 deg8). In support of our presumption we found that phosphorylation of D1 increased more in var2. Furthermore, the coexistence of var2 and stn8 was shown to recover the delay in degradation of D1, resulting in mitigation of the high vulnerability to photoinhibition of var2. Partial D1 cleavage fragments that depended on Deg proteases tended to increase, with concomitant accumulation of reactive oxygen species in the mutants lacking STN8. We inferred that the accelerated degradation of D1 in var2 stn8 presents a tradeoff in that it improved the repair of PSII but simultaneously enhanced oxidative stress. Together, these results suggest that PSII core phosphorylation prevents undesirable cleavage of D1 by Deg proteases, which causes cytotoxicity, thereby balancing efficient linear electron flow and photo‐oxidative damage. We propose that PSII core phosphorylation contributes to fine‐tuned degradation of D1.     

Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase.

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.     

Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase

3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis.     

Role of lysine 173 in heparin binding to heparin cofactor II

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits alpha-thrombin in a reaction that is dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site in HC by: (i) chemical modification with pyridoxal 5'-phosphate (PLP) in the absence and presence of heparin and dermatan sulfate; (ii) molecular modeling; and (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, and 348) were protected from modification by heparin and to a lesser extent by dermatan sulfate. Heparin-protected PLPHC retained both heparin cofactor and dermatan sulfate cofactor activity while dermatan sulfate-protected PLPHC retained some dermatan sulfate cofactor activity and little heparin cofactor activity. Molecular modeling studies revealed that Lys173 and Lys252 are within a region previously shown to contain residues involved in glycosaminoglycan binding. Lys343 and Lys348 are distant from this region, but protection by heparin and dermatan sulfate might result from a conformational change following glycosaminoglycan binding to the inhibitor. Site-directed mutagenesis of Lys173 and Lys343 was performed to further dissect the role of these two regions during HC-heparin and HC-dermatan sulfate interactions. The Lys343----Asn or Thr mutants had normal or only slightly reduced heparin or dermatan sulfate cofactor activity and eluted from heparin-Sepharose at the same ionic strength as native recombinant HC. However, the Lys173----Gln or Leu mutants had greatly reduced heparin cofactor activity and eluted from heparin-Sepharose at a significantly lower ionic strength than native recombinant HC but retained normal dermatan sulfate cofactor activity. Our results demonstrate that Lys173 is involved in the interaction of HC with heparin but not with dermatan sulfate, whereas Lys343 is not critical for HC binding to either glycosaminoglycan. These data provide further evidence for the determinants required for glycosaminoglycan binding to HC.     

Analysis of xylosyltransferase II binding to the anticoagulant heparin

The key enzymes in the biosynthetic pathway of glycosaminoglycan production are represented by the human xylosyltransferase I and its isoform II (XylT-I and XylT-II). The glycosaminoglycan heparin interacts with a variety of proteins, thereby regulating their activities, also those of xylosyltransferases. The identification of unknown amino acids responsible for heparin-binding of XylT-II was addressed in this study. Thus, six XylT-II fragments were designed as fusion proteins with MBP and we received soluble and purified MBP/XylT-II from Escherichia coli. Heparin-binding studies showed that all fragments bound with low affinity to heparin. Prolonging of XylT-II fragments did not account for a cooperative effect of multiple heparin-binding motifs and in turn for a stronger heparin-binding. Sequence alignment and surface polarity plot led to the identification of two highly positively charged Cardin-Weintraub motifs with surface accessibility, resulting in combination with short clusters of basic amino acids for strong heparin-binding of native xylosyltransferases.     

Interfacial versus homogeneous enzymatic cleavage of mandelonitrile by hydroxynitrile lyase in a biphasic system

The question of an interfacial versus a homogeneous reaction is carefully addressed for the enzymatic biphasic cleavage of mandelonitrile to benzaldehyde by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) (Hickel et al. [1999] Biotechnol Bioeng 36:425-436). Experimental evidence, including 1) the reaction ceases when the interface is populated by previously adsorbed denatured pa-Hnl, 2) the reaction continues even after washout of the bulk enzyme from the aqueous phase, 3) highly nonpolar organic solvents initially promote fast reaction kinetics that relatively quickly decay to zero product production, and 4) the reaction rate is nonlinear in the bulk enzyme concentration, provide robust grounds for an interfacial reaction. We also model enzymatic mandelonitrile cleavage assuming a homogeneous aqueous-phase reaction. The homogeneous reaction scheme does not simultaneously account for the experimental observations of a linear dependence of the reaction rate on organic/water interfacial area, no dependence on the aqueous-phase volume, and a nonlinear dependence on pa-Hnl aqueous concentration. Further, simple calculations demonstrate that the homogeneous reaction rate is at least three orders of magnitude slower than those observed by Hickel et al. (1999). We again conclude that enzyme adsorbed at the organic solvent/water interface primarily catalyzes the biphasic mandelonitrile cleavage reaction.     

Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase

The unusual enzyme, Gracilariopsis alpha-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of alpha-1,4-glucans via a beta-elimination reaction involving a covalent glycosyl-enzyme intermediate (Lee, S. S., Yu, S., and Withers, S. G. (2002) J. Am. Chem. Soc. 124, 4948-4949). The classical bell-shaped pH dependence of k(cat)/K(m) indicates two ionizable groups in the active site with apparent pK(a) values of 3.05 and 6.66. Br?nsted relationships of log k(cat) versus pK(a) and log(k(cat)/K(m)) versus pK(a) for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK(a) with low beta(lg) values of 0.32 and 0.33, respectively. The combination of these low beta(lg) values with large secondary deuterium kinetic isotope effects (k(H)/k(D) = 1.16 - 1.19) on the first step indicate a glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen at the transition state. Developed oxocarbenium ion character of the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin (K(i) = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-alpha-D-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large alpha-secondary deuterium kinetic isotope effect (k(H)/k(D) = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (k(H)/k(D) = 1.92) at C-2 confirm an E2 mechanism with strong E1 character for this second step. This considerable structural and mechanistic similarity with retaining alpha-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family.