108
T. Liu et al. / Process Biochemistry 48 (2013) 103–108
side chain (W490) to methyl side chain (A490) is the loss of
the hydrophobic interactions with +1 GlcNAc. By structure-based
sequence alignment, other chitinolytic enzymes including bacte-
in SpHex) (Fig. 6). This Trp is located in extended loops between
strand 7 and 8 of the (␣/)8-barrel catalytic domains whereas
the corresponding loop in HsHex, whose activity not affected by
substrate inhibition, is very short (Fig. 6) [27,28]. So, we believe
that the residue W490 determines the second substrate binding by
its bulky side chain.
[12] Schomburg I, Chang AJ, Hofmann O, Ebeling C, Ehrentreich F, Schomburg
D. BRENDA: a resource for enzyme data and metabolic information. Trends
Biochem Sci 2002;27:54–6.
[13] Reed MC, Lieb A, Nijhout HF. The biological significance of substrate inhibition:
a mechanism with diverse functions. Bioessays 2010;32:422–9.
[14] Andric P, Meyer AS, Jensen PA, Dam-Johansen K. Reactor design for minimizing
product inhibition during enzymatic lignocellulose hydrolysis: I. Significance
and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes.
Biotechnol Adv 2010;28:308–24.
[15] Chou YT, Yao SH, Czerwinski R, Fleming M, Krykbaev R, Xuan DJ, et al. Kinetic
characterization of recombinant human acidic mammalian chitinase. Biochem-
istry 2006;45:4444–54.
[16] Singh S, Gallagher R, Derrick PJ, Crout DHG. Glycosidase-catalyzed oligosac-
charide synthesis
– preparation of the N-acetylchitooligosaccharides
penta-N-acetylchitopentaose and hexa-N-acetylchitohexaose using the
-N-acetylhexosaminidase of Aspergillus oryzae. Tetrahedron: Asymmetry
1995;6:2803–10.
Acknowledgments
[17] Ryslava H, Kalendova A, Doubnerova V, Skocdopol P, Kumar V, Kukacka
Z, et al. Enzymatic characterization and molecular modeling of an evo-
lutionarily interesting fungal -N-acetylhexosaminidase. FEBS J 2011;278:
2469–84.
[18] Mayer C, Zechel DL, Reid SP, Warren RAJ, Withers SG. The E358S mutant of
Agrobacterium sp. -glucosidase is a greatly improved glycosynthase. FEBS Lett
2000;466:40–4.
[19] Mark BL, Wasney GA, Salo TJS, Khan AR, Cao ZM, Robbins PW, et al.
Structural and functional characterization of Streptomyces plicatus -N-
acetylhexosaminidase by comparative molecular modeling and site-directed
mutagenesis. J Biol Chem 1998;273:19618–24.
[20] Prag G, Papanikolau Y, Tavlas G, Vorgias CE, Petratos K, Oppenheim AB.
Structures of chitobiase mutants complexed with the substrate di-N-acetyl-
d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539
and glutamate 540. J Mol Biol 2000;300:611–7.
The authors acknowledge the financial support provided by
the National Key Project for Basic Research (2010CB126100),
the National Natural Science Foundation of China (31070715,
31101671), the National High Technology Research and Devel-
opment Program of China (2011AA10A204), the National Key
Technology R&D Program (2011BAE06B05), the Fundamen-
tal Research Funds for the Central Universities (DUT11ZD113,
DUT11RC(3)73).
References
[21] Manuel SGA, Ragunath C, Sait HBR, Izano EA, Kaplan JB, Ramasubbu N. Role
of active-site residues of dispersin B, a biofilm-releasing -hexosaminidase
[1] Tharanathan RN, Kittur FS. Chitin – the undisputed biomolecule of great poten-
tial. Crit Rev Food Sci 2003;43:61–87.
[2] Tamai Y, Miyatake K, Okamoto Y, Takamori Y, Sakamoto K, Minami S. Enhanced
healing of cartilaginous injuries by N-acetyl-d-glucosamine and glucuronic
acid. Carbohyd Polym 2003;54:251–62.
[3] Salvatore S, Heuschkel R, Tomlin S, Davies SE, Edwards S, Walker-Smith JA, et al.
A pilot study of N-acetyl glucosamine, a nutritional substrate for glycosamino-
glycan synthesis, in paediatric chronic inflammatory bowel disease. Aliment
Pharmacol Ther 2000;14:1567–79.
[4] Chen JK, Shen CR, Liu CL. N-Acetylglucosamine: production and applications.
Mar Drugs 2010;8:2493–516.
[5] Bissett DL, Robinson LR, Raleigh PS, Miyamoto K, Hakozaki T, Li J, et al. Reduction
in the appearance of facial hyperpigmentation by topical N-acetyl glucosamine.
J Cosmet Dermatol 2007;6:20–6.
[6] Binod P, Sandhya C, Suma P, Szakacs G, Pandey A. Fungal biosynthesis of endo-
chitinase and chitobiase in solid state fermentation and their application for the
production of N-acetyl-d-glucosamine from colloidal chitin. Bioresour Technol
2007;98:2742–8.
[7] Dahiya N, Tewari R, Hoondal GS. Biotechnological aspects of chitinolytic
enzymes: a review. Appl Microbiol Biotechnol 2006;71:773–82.
[8] Sashiwa H, Fujishima S, Yamano N, Kawasaki N, Nakayama A, Muraki E, et al.
Production of N-acetyl-d-glucosamine from -chitin by enzymatic hydrolysis.
Chem Lett 2001:308–9.
[9] Cederkvist FH, Parmer MP, Varum KM, Eijsink VGH, Sorlie M. Inhibition of a
family 18 chitinase by chitooligosaccharides. Carbohyd Polym 2008;74:41–9.
[10] Yang Q, Liu T, Liu FY, Qu MB, Qian XH. A novel -N-acetyl-d-hexosaminidase
from the insect Ostrinia furnacalis (Guenee). FEBS J 2008;275:5690–702.
[11] Liu T, Liu FY, Yang Q, Yang J. Expression, purification and characterization of the
chitinolytic -N-acetyl-d-hexosaminidase from the insect Ostrinia furnacalis.
Protein Expr Purif 2009;68:99–103.
from
a periodontal pathogen, in substrate hydrolysis. FEBS J 2007;274:
5987–99.
[22] Liu T, Zhang HT, Liu FY, Wu QY, Shen X, Yang Q. Structural determinants of
an insect -N-acetyl-d-hexosaminidase specialized as a chitinolytic enzyme. J
Biol Chem 2011;286:4049–58.
[23] Leonard R, Rendic D, Rabouille C, Wilson IBH, Preat T, Altmann F. The Drosophila
fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan
processing. J Biol Chem 2006;281:4867–75.
[24] Tews I, Perrakis A, Oppenheim A, Dauter Z, Wilson KS, Vorgias CE. Bacterial
chitobiase structure provides insight into catalytic mechanism and the basis of
Tay-Sachs disease. Nat Struct Biol 1996;3:638–48.
[25] Horn SJ, Sorlie M, Vaaje-Kolstad G, Norberg AL, Synstad B, Varum KM, et al.
Comparative studies of chitinases A, B and C from Serratia marcescens. Biocatal
Biotransform 2006;24:39–53.
[26] Mark BL, Vocadlo DJ, Knapp S, Triggs-Raine BL, Withers SG, James MNG.
Crystallographic evidence for substrate-assisted catalysis in a bacterial -
hexosaminidase. J Biol Chem 2001;276:10330–7.
[27] Maier T, Strater N, Schuette CG, Klingenstein R, Sandhoff K, Saenger W. The
X-ray crystal structure of human -hexosaminidase B provides new insights
into Sandhoff disease. J Mol Biol 2003;328:669–81.
[28] Mark BL, Mahuran DJ, Cherney MM, Zhao DL, Knapp S, James MNG.
Crystal structure of human -hexosaminidase B: understanding the molec-
ular basis of Sandhoff and Tay-Sachs disease.
1093–109.
J Mol Biol 2003;327:
[29] Lemieux MJ, Mark BL, Cherney MM, Withers SG, Mahuran DJ, James MNG.
Crystallographic structure of human -hexosaminidase A: interpretation of
Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis.
2006;359:913–29.
J Mol Biol