|
Chitinases (EC 3.2.1.14) hydrolyze the ?1,4 linkage of
chitin that consistsof straight chains of N-acetyl-D-glucosamine
residues linked ?1, 4. Variousorganisms including bacteria,
fungi, plants, and some vertebrates producechitinases, and
bacterial chitinases are thought to be important in thedigestion
of chitin for utilization as carbon and energy sources and serve
as animportant role in recycling chitin in nature. The C-
terminal domain of AeromonasHydrophila chitinase has been found
to have two 40 amino acid regions containingWXAXYWTXGXEP
consensus sequence. On the basis of homology search, chitinases(
U09139 and L42548), and cellulases (Z33876, P06565, and P06566)
contain one ortwo regions with consensus sequence. The strong
similarity of these amino acidsequences in chitinases and
cellulases suggests that this sequence is organizedinto domain
and performs specific function. In order to understand the
role of this consensus sequence, the C-terminaldomain of
Aeromonas Hydrophila chitinase with two WXAXYWTXGXEP
consensussequences has been isolated. The structural gene of the
89 residues C-terminaldomain of Aeromonas Hydrophila chitinase
(C89) was sequenced and expressed inE. coli BL21 from Dr. Ming-
Cheng Chang's group. The engineering strain ofE. coli JM105
carries the vector pGEX-5X-3 for the C-terminal domain.
Expressionof C89 was induced by addition of IPTG and was further
purified by GlutathioneSepharose 4B chromatography. The
separation of C89 from the glutathioneS-transferase was
accomplished by site specific proteolysis using factor Xa andwas
further purified by SP Sepharose chromatography. The
proteolytic product ofC89 named C52 was purified by Biogel P10
gel filtration chromatography. Based onSDS-polyacryamide gel
electrophoresis, the C89 and C52 of Aeromonas
Hydrophilachitinase were purified to be homogeneous. The
interaction of C89 with chitin and cellulose has been studied
byfluorescence and circular dichroism (CD) spectroscopy. Chitin
or cellulosebinding to C89 containing two WXAXYWTXGXEP consensus
sequences is accompanied bya large quenching of tryptophan
fluorescence, allowing the measurement ofequilibrium constants
for chitin-C89 (Kd= 6.5-22.2 潒) and cellulose-C89complexes
(Kd = 24.2-49 潒) with a 1:1 stoichiometry. On the contrary, C52
witha WXAXYWTXGXEP consensus sequence can not bind with chitin
and cellulose. Theseresults indicate that C89 binds chitin
tighter than cellulose, and an optimumsize of tetrameric
saccharides is required for binding. This may explain why
themain product of chitin cleavage is diacetylchitobiose. CD
analysis of C89 alone in PBS buffer at pH 7.4 shows 25.2% ?
helix, 6.5%?structure, and 68.3% coil, and the observed
structure is similar to thecalculated structure by GGBSM method.
N, N', N", N"'-Tetraacetylchitotetraosebinding increases the
structure of C89, changing the CD analysis to 37.9%?helix, 8.1%
?structure, and 54% coil. The binding of chitin to C-
terminaldomain of Aeromonas Hydrophila chitinase induces the
helical formation from coilon C89, and the conformational change
of C89 on chitin-binding may playimportant role for interaction
between chitin and bacterial chitinasescontaining WXAXYWTXGXEP
consensus sequence.
|