Crystallization of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex

Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain "pathogenesis-related" proteins from plants, but not to hen egg-white or phage T4 lysozyme. To investigate the atomic details of the substrate specificity and the cause for hevamine's low pH optimum (pH 4.0), we have crystallized two hevamine isozymes as a first step towards a high-resolution X-ray structure determination. Suitable crystals were obtained at room temperature from hanging drop experiments by vapor diffusion against 1.7 M to 3.4 M-NaCl (pH 5.0 to 9.0) for the major isozyme, and by vapor diffusion against 2.5 M to 4.3 M-NaCl (pH 5.0 to 8.0) for the minor one. Both isozymes give the same crystal morphology and space group. Their space group is P2(1)2(1)2(1) with cell dimensions a = 82.3 A, b = 58.1 A and c = 52.5 A (1 A = 0.1 nm). The crystals diffract to at least 2.0 A resolution.     

Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis

The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore. Products were separated by HPLC and the amount of product was calculated by peak integration. Penta-N-acetylglucosamine (penta-nag) and hexa-N-acetylglucosamine (hexa-nag) were used as substrates. Hexa-nag was more efficiently converted than penta-nag, which is an indication that hevamine has at least six sugar binding sites in the active site. Tetra-N-acetylglucosamine (tetra-nag) and allosamidin were tested as inhibitors. Allosamidin was found to be a competitive inhibitor with a K(i) of 3.1 microM. Under the conditions tested, tetra-nag did not inhibit hevamine.     

Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis.

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower k(cat) and a slightly higher K(m) than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with approximately 2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower k(cat) and an approximately twofold higher K(m) than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis.     

Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex

Several chitin-binding proteins were isolated from the bottom fraction of Hevea brasiliensis (Müll.) Arg. latex. One of these chitin-binding proteins is hevein, a small monomeric protein which strongly resembles the lectin from stinging nettle (Urtica dioica L.). Like the latter, hevein showed strong antifungal activity against several fungi in vitro. The possible involvement of this protein in the defense against invasion by potentially pathogenic fungi is discussed.Abbreviations FPLC fast protein liquid chromatography - M_r apparent molecular mass - SDS-PAGE Sodium dodecyl sulp-hatepolyacrylamide gel electrophoresis - UDA Urtica dioica agglutinin - WGA Wheat-germ agglutinin This work is supported in part by NIH grant and grants of the National Fund for Scientific Research (Belgium): W.J.P. is senior research associate, and W.F.B. senior research assistant of this fund. J.V.P. receives a fellowship of the Belgian Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw.     

The isolation and composition of helical protein microfibrils from Hevea brasiliensis latex

1. The microfibrils contained within the lutoid particles of Hevea brasiliensis latex obtained from young tissue have been isolated by methods based on low-speed centrifugation, isoelectric precipitation and gel filtration. 2. The isolated microfibrils behave as a single protein having an isoelectric point of about 4 as determined by paper electrophoresis. 3. The only components so far detected in the microfibrils are protein and possibly carbohydrate; nucleic acid appears to be absent. 4. The amino acid composition of the microfibril protein shows no unusual features. 5. In latex from the more mature laticiferous tissues of H. brasiliensis, the lutoid particles appear to be devoid of microfibrils or their protein decomposition products.     

Hevains: Serine-centred proteases from the latex of Hevea brasiliensis

Hevains b and l, isolated respectively from the serum and lutoids of freeze-dried latex from Hevea brasiliensis, were purified to homogeneity and compared with hevain a from commercial, ammonia-treated latex. The M_rs of hevains a and b are 69 000 and 58 000, respectively, and both exist in several charged forms. The amino acid compositions of the two enzymes differ significantly, but the reactivities to a variety of ester and protein substrates are similar, as are the pH optima. Hevain l is a distinct protease of M_r 80 000 and unique amino acid composition. It displays esterolytic activity and will digest insulin B chain, but is not proteolytic to azocollagen, azocasein, bovine serum albumen or haemoglobin. The activities of all three enzymes are dependent on the presence of serine and histidine residues.     

Polyphenol oxidases from latex of Hevea brasiliensis: purification and characterization

Polyphenol oxidase (PPO) was isolated from the B-serum obtained after repetitive freeze-thawing of the bottom fraction isolated from ultracentrifuged fresh latex. The B-serum was subjected to acetone precipitation and CM-Sepharose chromatography, affording two PPOs, PPO-I and PPO-II, which, upon SDS-PAGE, were 32 and 34 kDa, respectively. Both PPOs possessed the same pI (9.2), optimum pH (7) and optimum temperature (35-45 degrees C). They are stable up to 60 degrees C and active at broad pH ranges from 4-9. The K(m) values of PPO-I for dopamine, L-dopa and catechol as substrates are 2.08, 8.33 and 9.09 mM, while those for PPO-II are 2.12, 4.76 and 7.14 mM, respectively. Among various PPO inhibitors tested, 4-hexylresorcinol was the most potent. Anionic detergents were among the most effective activators of the enzymes, while cationic and nonionic detergents showed little and no effect on the PPO activities, respectively.     

The formation of 5-phosphomevalonate by mevalonate kinase in Hevea brasiliensis latex

1. Evidence has been produced for the formation of 5-phosphomevalonate from potassium dl-mevalonate by the latex of Hevea brasiliensis and by reconstituted freeze-dried serum obtained from this latex. 2. The enzyme, mevalonate kinase, catalysing the formation of 5-phosphomevalonate from potassium dl-mevalonate and ATP has been partially purified. 3. 5-Phosphomevalonate formed by the purified mevalonate kinase from potassium [2-(14)C]mevalonate has been shown to be incorporated by latex into rubber to about 2.4 times the extent of dl-mevalonate. 4. The enzyme can utilize inosine triphosphate as effectively as adenosine triphosphate as a phosphate donor and is also slightly active with uridine triphosphate. 5. The enzyme was fairly stable to a range of pH values and temperatures, the activity being optimum at pH7.5 and 60-70 degrees . The energy of activation was 10.7kcal./mole. The K(m) values were 0.13mm for potassium dl-mevalonate and 2.0mm for ATP at 30 degrees . 6. The enzyme required the presence of Mn(2+) (1mm) for maximum activity; this could be replaced by Mg(2+) (4mm), which was less effective, and by Ca(2+), which was far less effective. 6. Although the enzyme did not require cysteine or reduced glutathione for activation in aerobic conditions, it was inhibited by reagents known to react with thiol groups.     

Electron transport in the membrane of lutoids from the latex of Hevea brasiliensis.

1. An antimycin-insensitive NADH-cytochrome c oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex of Hevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH. 2. Two beta-type cytochromes are present in the membranes. Cytochrome beta563 is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 degrees K. A second cytochrome, cytochrome beta561, seems to be reducible by hydrosulfite only. 3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochrome P-450 could not be demonstrated. 4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis

We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.     

Demonstration of a pyrophosphate-fructose 6-phosphate 1-phosphotransferase in the latex from Hevea brasiliensis

A pyrophosphate: fructose-6-phosphate 1-phosphotransferase activity (EC 2.7.1.90) has been characterized in cytosol from Hevea brasiliensis latex. It is Mg+ dependent enzyme, and the cation has an optimal effect between 2.5 to 3 mM for a concentration of 1 mM of pyrophosphate and 10 mM of fructose-6-phosphate. It is activated by catalytic content of fructose-2,6-diphosphate. Its potential activity is higher than 40% of that of ATP dependent phosphofructokinase (EC 2.7.1.11). Its optimum pH is between 7.5-7.6; then, the enzyme affinity is 0.3 mM for pyrophosphate and 3.5 mM for fructose-6-phosphate. It is suggested that the transferase plays a role in the pyrophosphate metabolism and the increasing of the energetic efficiency of glycolysis and so takes a significant part in the biochemical mechanisms involved in the latex yield.