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Inhibiting effects of sulfated steroids from marine sponges of Halichondriidae family: halistanol sulfate, topsentiasterol sulfate D and chlorotopsentiasterol sulfate D were investigated on three different types of enzymes degrading polysaccharides of brown algae: endo-1,3-β-d-glucanase GFA, fucoidan hydrolase FFA2 and bifunctional alginate lyase ALFA3 from marine bacterium Formosa algae KMM 3553 T , inhabiting thalli of brown alga Fucus evanescens. This is the first research, devoted to influence of a marine natural compound on three functionally related enzymes that make up the complex of enzymes, necessary to degrade unique carbohydrate components of brown algae. Alginic acid, 1,3-β-D-glucan (laminaran) and fucoidan jointly constitute practically all carbohydrate biomass of brown algae, so enzymes, able to degrade such polysaccharides, are crucial for digesting brown algae biomass as well as for organisms surviving and proliferating on brown algae thalli. Halistanol sulfate irreversibly inhibited native endo-1,3-β-D-glucanases of marine mollusks, but reversibly competitively inhibited recombinant endo-1,3-β-d-glucanase GFA. This fact indicates that there are significant structural differences between the enzymes of practically the same specificity. For alginate lyase and fucoidan hydrolase halistanol sulfate was irreversible inhibitor. Topsentiasterol sulfate D was less active inhibitor whereas chlorotopsentiasterol sulfate D was the strongest inhibitor of enzymes under the study. Chlorotopsentiasterol sulfate D caused 98% irreversible inhibition of GFA. Chlorotopsentiasterol sulfate D also caused reversible and 100% inhibition of ALFA3, which is unusual for reversible inhibitors. Inhibition of FFA2 was complete and irreversible in all cases., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD.

Bacterial heparinases that cleave heparan sulfate (HS) and heparin are widely used to generate low-molecular-weight heparins (LMWHs) and to structurally and functionally characterise heparin and HS biomolecules. We provide novel insights into the substrate specificity of heparinase II from two different bacteria: Pedobacter heparinus (formerly Flavobacterium heparinum) and Bacteroides eggerthii. The activity towards various well-defined HS oligosaccharides was investigated by (1) H NMR spectroscopy; this revealed distinct specificities for the two heparinases. Heparinase II from P. heparinus appears to be more active and displays a broader substrate specificity than B. eggerthii heparinase II. Furthermore, HS di- and tetrasaccharides inhibited B. eggerthii heparinase II activity. A better understanding of heparinase substrate specificity will contribute to the production of homogenous LMWHs, provide better characterisation of heparin and HS and assist therapeutic applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Heparanase, an endo-β-glucuronidase cleaving heparan sulfate (HS) chains at cell surfaces and in the extracellular matrix (ECM), is involved in angiogenesis, tumor progression and metastasis as well as in inflammation and kidney dysfunction. Therefore, heparanase is considered a promising therapeutic target and diagnostic marker. Recently, we have developed a simple, rapid, fully automatable fluorimetric activity assay using the synthetic sulfated pentasaccharide fondaparinux as substrate and bacterial heparinase II (HEP-II) instead of human heparanase (hHEP). The aim of this study was to evaluate this assay for inhibitor testing as well as to check whether the assay principle is applicable to measure the activity and inhibition, respectively, of the actual target enzyme hHEP. Besides the known hHEP inhibitor suramin and the antiinflammatory and antimetastatic PS3, two series of β-1,3-glucan sulfates differing in their chain length and degree of sulfation, further semisynthetic sulfated glycans, and two sulfated polysaccharides isolated from algae were included to examine structure-activity relationships. The inhibitory activity of sulfated glycans showed to be greatly dependent on both their degree of sulfation and their basic glycan structure, but independent of their molecular size. The β-1,3-glucan sulfates were superior to suramin as well as to the other glycans with similar degree of sulfations. The most active inhibitor was found to be the β-1,3-glucan sulfate PS3 (IC₅₀=0.017 μM). By using hHEP instead of HEP-II comparable results were obtained. With an IC₅₀ being about 160 times lower than that of suramin, PS3 exhibited again the strongest inhibitory effects. Inhibition of hHEP may therefore contribute to the potent antiinflammatory and antimetastatic activities of PS3 in vivo. In conclusion, the fluorimetric hHEP activity assay proved to be a simple, fully automatable tool for testing potential inhibitors. In case of HS mimetic inhibitors, the assay variant with HEP-II may provide a fast and inexpensive option for initial screening purposes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.

Heparanase-1 (HPSE-1) is an endoglycosidase that cleaves heparan sulfate. The physiological functions of HPSE-1 include embryo development, hair growth, wound healing, tumor growth, angiogenesis, metastasis, and inflammation. HPSE-1 expression was found to increase temporarily in the rat ventral prostate (VP) after castration. The promoter region of the Hpse-1 gene has estrogen-responsive elements, suggesting that the gene is regulated by estrogens. In this study, we investigated the expression of HPSE-1 in the VP of 90-day-old rats after neonatal exposure to a high dose of 17β-estradiol. HPSE-1 was not found by immunohistochemistry in the epithelium of estrogenized animals. To determine whether inhibition of Hpse-1 expression in the epithelium was due to pre- or post-transcriptional regulation, epithelial cells were isolated by centrifugation in Percoll gradient and the presence of Hpse-1 mRNA was investigated by RT-PCR. Hpse-1 mRNA was not detected in the estrogenized animals. Considering that Hpse-1 transcription could be inhibited by DNA methylation, we used the methylation-sensitive restriction enzyme HpaII and PCR to show that a single CCGG site at position +185 was more frequently methylated in the epithelium of estrogenized than in control animals. Immunohistochemistry for 5-methylcytidine revealed that the epithelial cell nuclei in estrogenized animals were heavily methylated. These results suggest that Hpse-1 expression was blocked in the epithelial cells of the VP, by estrogen imprinting by a pre-transcriptional mechanism involving DNA methylation.

Bacterial hyaluronan lyases (Hyal) degrade hyaluronan, an important component of the extracellular matrix, and are involved in microbial spread. Hyal inhibitors may serve as tools to study the role of the enzyme, its substrates and products in the course of bacterial infections. Moreover, such enzyme inhibitors are potential candidates for antibacterial combination therapy. Based on crystal structures of Streptococcus pneumoniae Hyal in complex with a hexasaccharide substrate and with different inhibitors, 1-acylated benzimidazole-2-thiones and benzoxazole-2-thiones were derived as new leads for the inhibition of Streptococcus agalactiae strain 4755 Hyal. Structure-based optimization led to N-(3-phenylpropionyl)benzoxazole-2-thione, one of the most potent compounds known to date (IC(50) values: 24 μM at pH 7.4, 15 μM at pH 5). Among the 27 new derivatives, other N-acylated benzimidazoles and benzoxazoles are just as active at pH 7.4, but not at pH 5. The results support a binding mode characterized by interactions with residues in the catalytic site and with a hydrophobic patch., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

To characterize the alginate lyase produced by rhizosphere Streptomyces, Streptomyces sp. A5 was isolated from banana rhizosphere, and its extracellular lyase was purified to an electrophoretically homogeneous state. The lyase has a molecular mass of 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and pH were 37 degrees C and pH 7.5, respectively. Ninety-two percent of the activity was lost after incubation at 70 degrees C and pH 7.5 for 20 min. The enzyme was inhibited by 0.05 M SDS and 2 mM Hg2+, Cu2+, and Fe3+, but EDTA enhanced the enzyme activity. The Km value of the lyase was 0.13 mg mL-1 with the substrate sodium alginate. The lyase had substrate specificity for polyguluronate units in the alginate molecules. The alginate oligomers prepared by the lyase show growth-promoting activity on the roots of banana plantlets. These results indicated that the encapsulation method using alginate microbeads to inoculate beneficial streptomycete strains might be beneficial to the root growth of banana plantlets.

The bacterial hyaluronan lyases (Hyals) that degrade hyaluronan, an important component of the extracellular matrix, are involved in microbial spread. Inhibitors of these enzymes are essential in investigation of the role of hyaluronan and Hyal in bacterial infections and constitute a new class of antibiotics against Hyal-producing bacteria. Recently, we identified 1,3-diacetylbenzimidazole-2-thione and related molecules as inhibitors of streptococcal Hyal. One of such compounds, 1-decyl-2-(4-sulfamoyloxyphenyl)-1-indol-6-yl sulfamate, was co-crystallized in a complex with Streptococcus pneumoniae Hyal and its structure elucidated. The resultant X-ray structure demonstrates that this inhibitor fits in the enzymatic active site via interactions resembling the binding mode of the natural hyaluronan substrate. X-ray structural analysis also indicates binding interactions with the catalytic residues and those of a catalytically essential hydrophobic patch. An IC50 value of 11 microM for Hyal from Streptococcus agalactiae (strain 4755) qualifies this phenylindole compound as one of the most potent Hyal inhibitors known to date. The structural data suggested a similar binding mode for N-(3-phenylpropionyl)-benzoxazole-2-thione. This new compound's inhibitory properties were confirmed resulting in discovery of yet another Hyal inhibitor (IC50 of 15 microM). These benzoxazole-2-thiones constitute a new class of inhibitors of bacterial Hyals and are well suited for further optimization of their selectivity, potency, and pharmacokinetic properties.

Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.

The effect of triterpenes and flavonoids on the activity of several hyaluronic acid-splitting enzymes was investigated. Studies showed that the inhibitory effect of the triterpenes glycyrrhizin and glycyrrhetinic acid is dependent on the source of hyaluronate lyase. Hyaluronate lyase from Streptococcus agalactiae (Hyal B) and recombinant hyaluronate lyase from Streptococcus agalactiae (rHyal B) demonstrated strongest inhibition. In contrast, hyaluronate lyases from Streptomyces hyalurolyticus (Hyal S), Streptococcus equisimilis (Hyal C) and hyaluronidase from bovine testis (Dase) showed only reduced inhibition action. A non-competitive dead end inhibition with Ki=3.1+/-1.8x10(-6) mol/mL and Kii=6.7+/-2.4x10(-6) mol/mL was found for glycyrrhizin on recombinant hyaluronate lyase from Streptococcus agalactiae. The inhibitory effect of flavonoids on Hyal B, rHyal B and Dase was determined depending on the number of hydroxyl groups and side chain substituents in the molecule. Flavonoids with many hydroxyl groups inhibited hyaluronate lyase stronger than those with only a few. Native hyaluronate lyase (Hyal B) showed a more extensive inhibition than the recombinant protein (rHyal B). Accordingly, the inhibition by triterpenes and flavonoids is presumably specific for each hyaluronic acid (HA)-splitting enzyme.

The crystal structure of alginate (poly alpha-l-guluronate) lyase from Corynebacterium sp. (ALY-1) was determined at 1.2A resolution using the MAD method and bromide ions. The structure of ALY-1 is abundant in beta-strands and has a deep cleft, similar to the jellyroll beta-sandwich found in 1,3-1,4-beta-glucanase. The structure suggests that alginate molecules may penetrate into the cleft to interact with the catalytic site of ALY-1. The reported crystal structure of another type of alginate lyase, A1-III, differs from that of ALY-1 in that it consists almost entirely of alpha-helical structure. Nevertheless, the putative catalytic residues in both enzymes are positioned in space in nearly identical arrangements. This finding suggests that both alginate lyases may have evolved through convergent evolution.

A thio-linked disaccharide based on the structure of the glycosaminoglycan chondroitin was synthesized as a potential inhibitor of chondroitin AC lyase from Flavobacterium heparinum for structural analysis of the active site. Instead it was found to be a slow substrate, thereby demonstrating that lyases, in contrast to glycosidases, can cleave thioglycoside links between sugars.

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. Brønsted 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.

Enzyme activity measurement showed that L-ascorbic acid (vitamin C (Vc)) competitively inhibits the hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. The complex crystal structure of this enzyme with Vc was determined at 2.0 A resolution. One Vc molecule was found to bind to the active site of the enzyme. The Vc carboxyl group provides the negative charges that lead the molecule into the highly positively charged cleft of the enzyme. The Vc ring system forms hydrophobic interactions with the side chain of Trp-292, which is one of the aromatic patch residues of this enzyme responsible for the selection of the cleavage sites on the substrate chain. The binding of Vc inhibits the substrate binding at hyaluronan 1, 2, and 3 (HA1, HA2, and HA3) catalytic positions. The high concentration of Vc in human tissues probably provides a low level of natural resistance to the pneumococcal invasion. This is the first time that Vc the direct inhibition on the bacterial "spreading factor" was reported, and Vc is also the first chemical that has been shown experimentally to have an inhibitory effect on bacterial hyaluronate lyase. These studies also highlight the possible structural requirement for the design of a stronger inhibitor of bacterial hyaluronate lyase.

The syntheses of methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside (1) and methyl alpha-D-xylo-hex-4-ulopyranosyl-(1-->4)-alpha-D-galactopyranoside (4) are reported. The keto-disaccharide 4 is of interest in our design, synthesis, and study of pectate lyase inhibitors. The key step in the syntheses was the high-yielding, stereospecific formation of methyl 4,6-O-benzylidene-2',3'-di-O-benzyl-alpha-D-glucopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (15), which was accomplished by reacting 2,3-di-O-benzyl-4,6-O-benzylidene-D-glucopyranosyl trichloroacetimidate (10) with methyl 2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (14) in the presence of a catalytic amount of tert-butyldimethylsilyl trifluoromethane sulfonate (TMSOTF). Compound 15 was either hydrogenolyzed to yield disaccharide 1 or treated with NaBH3CN-HCl in 1:1 tetrahydrofuran-ether to yield methyl 2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (2). The free 4'-OH of compound 2 was oxidized to a carbonyl group by a Swern oxidation, and the protecting groups were removed by hydrogenolysis to yield keto-disaccharide 4. These synthetic pathways were simple, yet high yielding.

The kinetic properties and active site amino acids of alpha-1,4-glucan lyase from the marine red macroalga Gracilariopsis sp. were examined. Using 1H NMR spectroscopy the alpha-1,4-glucan lyase was found to degrade alpha- and beta-maltose at different rates. The effect of pH on the kinetic constants suggested the presence of two catalytically important amino acids in the active site with pKa values of 3.5 and 6.2. The former indicated the presence of an ionised aspartate or glutamate residue in the active site. This was tested using the carboxyl specific reagent EDAC, which inhibited enzyme activity in a time dependent manner when an external nucleophile was added. No protection against the inactivation was obtained by addition of amylopectin, maltitol or 1-deoxinojirimycin. Inactivation decreased Vmax over 2.5-fold with little effect on Km which supports the direct involvement of a carboxyl group in catalysis.

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.

In the presence of a known colon carcinogen, 1,2-dimethyl hydrazine (DMH), the activity of beta-glucuronidase was found to be significantly increased in the distal colon, distal intestine, liver and colon contents and the activity of mucinase was increased in both the colon and fecal contents when compared to control rats. Chilli (Capsicum annum L., Solanaceae) administration also showed an increase when compared to control rats, whereas supplementation with cumin (Cuminum cyminum L., Apiaceae) and black pepper (Piper nigrum L., Piperaceae) in the presence of DMH, showed more or less similar values as that of the control rats. The increase in beta-glucuronidase activity may increase the hydrolysis of glucuronide conjugates, liberating the toxins, while the increase in mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Thus cumin and black pepper may protect the colon by decreasing the activity of beta-glucuronidase and mucinase. Histopathological studies also showed lesser infiltration into the submucosa, fewer papillae and lesser changes in the cytoplasm of the cells in the colon in cumin and black pepper groups when compared to the DMH and chilli treated animals.

In Erwinia chrysanthemi 3937, pectate lyase activity mainly results from the cumulative action of five major isoenzymes, PelA to PelE. Comparison of their amino acid sequences revealed two families, PelB-C and PelA-D-E. Molecular cloning permitted expression of the different pel genes in Escherichia coli and the isolation of each Pel independently from the other isoenzymes. We used similar experimental conditions to overproduce and purify the five Pels in a one-step chromatography method. We analyzed some of the basic enzymatic properties of these five isoenzymes. PelA has a low specific activity compared to the other four enzymes. PelB and PelC have a high affinity for their substrate: about 10-fold higher than the enzymes of the PelA-D-E group. The optimum pH is more alkaline for PelB and PelC (about 9.2) than for PelA, PelD, and PelE (from 8 to 8.8). Below pH 7, activity was negligible for PelB and PelC, while PelA, PelD, and PelE retained 25 to 30% of their activities. The temperature optima were determined to be 50 degrees C for PelD and PelE, 55 degrees C for PelA, and 60 degrees C for PelB and PelC. Enzymes of the PelB-C group are more stable than those of the PelA-D-E group. Use of substrates presenting various degrees of methylation revealed that PelA, PelD, and PelE are active only for very low levels of methylation, while PelB and PelC are more active on partially methylated pectins (up to 22% for PelC and up to 45% for PelB). Pectate lyases have an absolute requirement for Ca2+ ions. For the five isoenzymes, maximal activity was obtained at a Ca2+ concentration of 0.1 mM. None of the tested cations (Ba2+, Co2+, Cu2+, Mg2+, Mn2+, Sr2+, Zn2+) can substitute for Ca2+. At a high concentration (1 mM), most of the divalent cations inhibited pectate lyase activity. In addition, we demonstrated that two compounds present in plant tissues, epicatechin and salicylic acid, inhibit the pectate lyases at a concentration of 0.2 mM.

Hyaluronate lyase produced by group B streptococci (GBS) degrades hyaluronan completely to unsaturated disaccharide units and also cleaves unsulfated regions of chondroitin sulfate. The enzyme is rapidly inactivated by diethyl pyrocarbonate and enzymatic activity is restored by treatment with hydroxylamine, suggesting that a histidine residue is present in the active site. Amino acid sequence comparisons of GBS hyaluronate lyase and four other related enzymes revealed that one of the 16 histidine residues of the enzyme (His-479) is present in a highly conserved region. Conversion of His-479 to a glycine by site-directed mutagenesis resulted in a complete loss of enzymatic activity of the modified protein. We propose that His-479 is in the active site of GBS hyaluronate lyase and participates in the initial abstraction of hydrogen ions from the glucuronic acid residues of hyaluronan.

In view of the role that heparitinase and heparanase enzymes are thought to play in regulating the proliferation of smooth muscle cells, inhibitors of these enzymes may have therapeutic value in the treatment of vascular hyperplasia. Here we report that phosphorothioate oligodeoxynucleotides inhibit platelet heparitinase and related enzymes in vitro. The inhibition is greatly enhanced by the presence of a GGG motif in the oligonucleotide, and also increases with oligonucleotide for two phosphorothioate DNA 30-mers consisting solely of guanosine and thymidine nucleotides. Their inhibitory efficacy was greater when heparan sulphate was used as substrate.

Previous studies have shown the presence in faeces of sulphatases, sialidases, glycosidases, and proteases relevant to mucus degradation, but the relative role of these enzymes in the degradation of colonic mucus has been unclear. A total mucinase assay using 14C threonine biologically labelled human colonic mucin as substrate was therefore developed in this study. Faecal mucinase activity of a pooled normal faecal filtrate was capable of removing 80% of the 14C threonine label from mucin within eight hours incubation, but 20% remained intact despite prolonged incubation. The pH profile of mucinase activity is broad (pH 4.5-9.5) suggesting contribution from multiple enzymes. Mucinase activity was reduced by preincubation with 100 micrograms/ml chymostatin (82.8%), 0.5 mg/ml EDTA (91.6%), and 4 g/l bismuth subsalicylate (72.0%). All 55 faecal samples studied contained detectable mucinase activity, measured as dpm release/micrograms protein/hour, which was greater in samples from patients with ulcerative colitis (n = 17, median 52.7, interquartile range 32.9-66.9), than controls (n = 26, 34.4, 26.8-40.4, p < 0.02) or patients with Crohn's disease (n = 12, 35.5, 17.5-55.7, p < 0.05). There was, however, no significant difference in faecal mucinase activity between inactive and active ulcerative colitis. These results suggest that faecal mucinase activity is one factor contributing to the thin mucus layer in ulcerative colitis and represents a potential target for drug treatment.

Four antibiotics were tested against Erwinia causing soft rot of onion (Allium cepa var. aggregatum) of which streptomycin sulphate 90% and tetracycline hydrochloride 10% (streptocycline) recorded the maximum inhibition zone of 27.66 mm. In the enzyme studies the maximum inhibition of pectinlyase (PL), polygalacturonase (PG) and protopectinase production was recorded by the same antibiotic. The antibiotics have a significant influence on the production and activity of cell wall degrading enzymes produced by the plant pathogenic microorganisms. Garlic clove extract was equally effective in inhibiting the growth and enzyme production.

Pectate lyase (PL) from Colletotrichum gloeosporioides was purified to apparent homogeneity by hydrophobic interaction chromatography followed by isoelectric focusing. The purified preparation showed one band corresponding to 40 kD on sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the enzyme was 7.9, and the optimum pH for activity was 8.9. The purified PL efficiently macerated unripe avocado fruit wedges. In vitro translation of mRNA from an induced fungal culture revealed a 36-kD precursor polypeptide, which was precipitated with PL antibodies. The antibodies inhibited enzymatic activity and maceration ability on avocado wedges. Epicatechin, a flavan 3-ol present in the peel of unripe avocado fruit, had a Ki of 3.4 microM for inhibition of PL activity in vitro. At 20 micrograms/ml (68 microM), epicatechin reduced the enzyme's macerating ability by 64%. Since the flavan is present in unripe fruit at much higher concentrations (about 350 micrograms/g fresh weight) than the inhibitory concentrations, epicatechin may be involved in the resistance of unripe avocado fruits by inhibiting the PL activity of C. gloeosporioides.

Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.

The ability of activated T lymphocytes to penetrate the extracellular matrix and migrate to target tissues was found to be related to expression of a heparanase enzyme (Naparstek, Y., I. R. Cohen, Z. Fuks, and I. Vlodavsky. 1984. Nature (Lond.). 310:241-243; Savion, N., Z. Fuks, and I. Vlodavsky. 1984. J. Cell. Physiol. 118:169-176; Fridman, R., O. Lider, Y. Naparstek, Z. Fuks, I. Vlodavsky, and I. R. Cohen. 1987. J. Cell. Physiol. 130:85-92; Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We found previously that heparin molecules inhibited expression of T lymphocyte heparanase activity in vitro and in vivo, and administration of a low dose of heparin in mice inhibited lymphocyte traffic and delayed-type hypersensitivity reactions (Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We now report that treatment with commercial or chemically modified heparins at relatively low doses once daily (5 micrograms for mice and 20 micrograms for rats) led to inhibition of allograft rejection and the experimental autoimmune diseases adjuvant arthritis and experimental autoimmune encephalomyelitis. Higher doses of the heparins were less effective. The ability of chemically modified heparins to inhibit these immune reactions was associated with their ability to inhibit expression of T lymphocyte heparanase. There was no relationship to anticoagulant activity. Thus heparins devoid of anticoagulant activity can be effective in regulating immune reactions when used at appropriate doses.

Pectate lyase synthesis in the cells of Erwinia carotovora ELA 49 is induced by polypectate. This suggested that the Erwinia chromosomes carried a regulator gene responsible for negative regulation of the pectate lyase gene expression. In the present study the regulator gene controlling expression of one of the pectate lyase structural genes was cloned and designated as ptlA gene. For this purpose a genetic system with the tester plasmid pPc624 as the main element was constructed. The tester plasmid contained cat gene (resistance to chloramphenicol) controlled by the promotor of the ptlA gene cloned on vector pPD620. Plasmid pPC624 was maintained in the E. coli cells in a number of 1-2 copies and transferred resistance to chloramphenicol in concentrations up to 100 micrograms/ml to the cells. The E. carotovora cells containing pPC624 were sensitive to chloramphenicol in media containing no inductor (sodium polypectate). In media with the inductor they were resistant to chloramphenicol. Therefore, plasmid pPC624 proved to be a suitable system for testing the regulator gene product. The E. coli cells containing plasmid pPC624 were transformed by the hybrid Ptl+ plasmids identified in the clonotheque of the Erwinia DNA EcoRI fragments. The E. coli cotransformants were characterized by chloramphenicol sensitivity which provided a conclusion that the regulator ptlR gene controlling the ptlA gene expression was localized on the DNA EcoRI fragment (7.3 kb) containing the pectate lyase ptlA and ptlB genes. Deletion analysis showed that the investigated genes were localized in the EcoRI fragment (7.3 kb) of the E. carotovora chromosomal DNA in the following order: ptlA--ptlB--ptlR.(ABSTRACT TRUNCATED AT 250 WORDS)