Computing and Library Services - delivering an inspiring information environment

Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

Neustroev, Kirill N., Golubev, Alexander M, Sinnott, Michael L., Borriss, Rainer, Krah, Martin, Brumer III, Harry, Eneyskaya, Elena V., Shishlyannikov, Sergey, Shabalin1, Konstantin A., Peshechonov, Viacheslav T., Korolev, Vladimir G. and Kulminskaya, Anna A. (2006) Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants. Glycoconjugate Journal, 23 (7/8). pp. 501-511. ISSN 0282-0080

Metadata only available from this repository.


Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency values, k cat/K m, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1, 3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxside

Item Type: Article
Subjects: Q Science > Q Science (General)
Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
Schools: School of Applied Sciences
School of Applied Sciences > Biomolecular Sciences Research Centre
Related URLs:

1. Henrissat, B.: A classification of glycosyl hydrolases based on
amino acid sequence similarities, Biochem. J. 280, 309–16 (1991)
2. Michel, C., Chantalat, L., Duee, E., Barbeyron, T., Henrissat, B.,
Kloareg, B., Dideberg, O.: The κ-carrageenanase of P. carrageenovora
features a tunnel-shaped active site: a novelin the evolution of clan B glycoside hydrolases. Structure 9, 513–
25 (2001)
3. Planas, A.: Bacterial 1,3-1,4-β-glucanases: structure, function and
protein engineering. Biochim. Biophys. Acta 1543, 361–82 (2000)
4. Allouch, J., Jam, M., Helbert,W., Barbeyron, T., Kloareg, B., Henrissat,
B., Czjzek, M.: The three-dimensional structures of two β-
agarases. J. Biol. Chem. 278, 47171–80 (2003)
5. Johansson, P., Brumer, H., Baumann, M.J., Kalla, A.M., Henriksson,
H., Denman, S.E., Teeri, T.T., Jones, T.A.: Crystal Structures
of poplar xyloglucan endotransglycosylase reveal details of transglycosylation
acceptor binding. Plant. Cell. 16, 874–86 (2004)
6. Vocadlo, D.J., Davies, G.J., Laine, R., Withers, S.G.: catalysis by
hen egg-white lysozyme proceeds via a cavalent intermediate. Nature
412, 835–8 (2001)
7. Sinnott, M.L., Catalytic mechanisms of enzymatic glycosyl transfer.
Chem. Rev. 90, 1171–1202 (1990)
8. Krah, M., Misselwitz, R., Politz, O., Thomsen, K.K., Welfle,
H., Borriss, R.: The laminarinase from thermophilic eubacterium
Rhodothermus marinus. Conformation, stability, and identification
of active site carboxylic residues by site-directed mutagenesis. Eur.
J. Biochem. 257, 101–11 (1998)
9. Godfrey, T.: On comparison of key characteristics of industrial enzymes
by type and source. In: Godfrey, T., Reinchelt, J.(eds.) Industrial
Enzymology pp. 466. Macmillan, London (1983)
10. Jakeman, D.,Withers, S.G., Glycosynthases: newtools for oligosaccharide
synthesis. Trends Glycosc. Glycotechnol. 14, 13–25 (2002)
11. Mackenzie, L.F.,Wang, Q.,Warren, R.A.,Withers, S.G., Glycosynthases:
mutant glycosidases for oligosaccharide synthesis. J. Am.
Chem. Soc. 120, 5583–4 (1998)
12. Mayer, C., Zechel, D.L., Reid, S.R., Warren, A.J., Withers, S.G.:
The E358S mutant of Agrobacterium sp. β-glucosidase is a greatly
improved glycosynthase. FEBS Let. 466, 40–4 (2000)
13. Viladot, J.-L., Canals, F., Batllori, X., Planas, A.: Long-lived
glycosyl-enzyme intermediate mimic produced by formate reactivation
of a mutant endoglucanase lacking its catalytic nucleophile.
Biochem. J. 355, 79–86 (2001)
14. Williams, S.J., Withers, S.: Glycosyl fluorides in enzymatic reactions.
Carbohydr. Res. 327, 27–46 (2000)
15. Rivera, M.H., Lypez-Munguha, A., Soberyn, X., Saab-Rincyn, G.:
α-Amylase from Bacillus licheniformis mutants near to the catalytic
site: effects on hydrolytic and transglycosylation activity. Protein
Engineer 16, 505–14 (2003)
16. Matsui, I., Yoneda, S., Ishikawa, K., Miyairi, S., Fukui, S.,
Umeyama, H., Honda, K.: Roles of the aromatic residues conserved
in the active center of Saccharomycopsis α-amylase for
transglycosylation and hydrolysis activity. Biochem. 33, 451–8
17. Hansson, T., Kaper, T., van der Oost, J., de Vos,W.M., Aldercreutz,
P.: Improved oligosaccharide synthesis by protein engineering of
β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus.
Biotechnol. Bioengin. 73, 203–10 (2001)
18. Viladot, J.-L., Stone, B., Driguez, H., Planas, A.: Expeditious synthesis
of a new hexasaccharide using transglycosylation reaction
catalysed by Bacillus (13),(14)-β-D-glucan 4-glucanohydrolase.
Carbohydr. Res. 311, 95–9 (1998)
19. Viladot, J.-L., Moreau, V., Planas, A., Driguez, H.: Transglycosylation
activity of Bacillus 1,3-1,4-β-D-glucan 4-glucanohydrolase.
Enzymatic studies of alternate 1,3-1,4-β-D-glucooligosaccharides.
J. Chem. Soc., Perkin Trans 1, 2383–7 (1997)
20. Borriss, R., Krah, M., Brumer 3rd, H., Kerzhner, M.A.,
Elyakova, L.A., Ivanen, D.R., Eneyskaya, E.V., Shishlyannikov,
S.M., Shabalin, K.A., Neustroev, K.N.: Enzymatic synthesis of
4-methylumbelliferyl β-(1,3)-D-glucooligosaccharides–new substrates
for 1,3(4)-β-glucanase. Carbohydr. Res. 338, 1455–7 (2003)
21. Kataoka, K., Muta, T., Yamazaki, S., Takeshige, K.: Activation
of macrophages by linear (13)-β-D-glucans. Implications for the
recognition of fungi by innate immunity. J. Biol. Chem. 277, 36825–
31 (2002)
22. Lowe, E., Rice, P.,Ha T, LiC,Kelley, J., Ensley, H., Lopez-Perez, J.,
Kalbfleisch, J., Lowman, D., Margl, P., Browder, W.D., Williams,
A.: (1,3)-β-D-linked heptasaccharide is the unit ligand for glucan
pattern recognition receptors on human monocytes. Microbes. Infec.
3, 789–97 (2001)
23. Laemmli, U.K.: Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227, 680–5 (1970)
24. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J.: Protein
measurements with the Folin phenol reagent. J. Biol. Chem. 193,
265–75 (1951)
25. Ogawa, K., Tsurugi, J.,Watanabe,T.: The dependence of the conformation
of a (1,3)-β-D-glucan on chain-length in alkaline solution.
Carbohydr. Res. 29, 397–403 (1973)
26. Petersen, B.O., Krah, M., Duus, J.O., Thomsen, K.K.: A transglycosylating
1,3(4)-β-glucanase from Rhodothermus marinus NMR
analysis of enzyme reactions. Eur. J. Biochem. 267, 361–9 (2000)
27. Kulminskaya, A.A., Thomsen, K.K., Shabalin, K.A., Sidorenko,
I.A., Eneyskaya, E.V., Savel’ev, A.N., Neustroev, K.N.: Isolation,
enzymatic properties, and mode of action of an exo-1,3-β-glucanase
from Trichoderma viride. Eur. J. Biochem. 268, 6123–31 (2001)
28. Somogyi, M.: Notes on sugar determination. J. Biol. Chem. 195,
19–23 (1952)
29. Eneyskaya, E.V., Brumer 3rd H., Backinowsky, L.V., Ivanen, D.R.,
Kulminskaya, A.A., Shabalin, K.A., Neustroev, K.N.: Enzymatic
synthesis of β-xylanase substrates: Transglycosylation reactions of
the β-xylosidase from Aspergillus sp., Carbohydr. Res. 338, 313–25
30. Malet, C., Planas, A.: Mechanism of Bacillus 1,3;1,4-β-Dglucan
4-glucanohydrolases: kinetics and pH studies with 4-
methylumbelliferyl β-D-glucan oligosaccharides. Biochem. 36,
13838–48 (1997)
31. Fujimoto, H., Isomura, M., Miyazaki, T., Matsuo, I., Walton, R.,
Sakakibara, T., Ajisaka, K.: Enzymatic syntheses of GlcNAc β-1-
2Man and Gal-β-1-4GlcNAc β-1-2Man as components of complex
type sugar chains. Glycoconj. J. 14, 75–80 (1997)
32. Pitson, S.M., Seviour, R.J., McDougall, B.M., Woodward, J.R.,
Stone, B.A.: Purification and characterization of three extracellular
(13)-β-D-glucan glucohydrolases from the filamentous fungus
Acremonium persicinum. Biochem. J. 308, 733–41 (1995)
33. Kraulis, P.J.: MOLSCRIPT: a program to produce both detailed
and schematic plots of protein structures. J. Appl. Crystallog. 24,
946–50 (1991)
34. Christensen, U., Olsen, K., Stoffer, B.B., Svensson, B.: Substrate
binding mechanism of Glu180 − >Gln, Asp176 ->Asn and wildtype
glucoamylases from Aspergillus niger. Biochem. 35, 15009–
18 (1996)
35. Christensen, T., Stoffer, B.B., Svensson, B., Christensen, U.: Some
details of the reaction mechanism of glucoamylase from Aspergillus
niger - Kinetic and structural studies on Trp52 − > Phe and Trp
317 − >Phe mutants. Eur. J. Biochem. 250, 638–45 (1997) insight

Depositing User: Sara Taylor
Date Deposited: 14 Mar 2008 15:18
Last Modified: 20 Oct 2008 10:46


Downloads per month over past year

Repository Staff Only: item control page

View Item View Item

University of Huddersfield, Queensgate, Huddersfield, HD1 3DH Copyright and Disclaimer All rights reserved ©