Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic <Emphasis Type="Italic">Kocuria varians</Emphasis> Alpha-Amylase |
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Authors: | Rui Yamaguchi Tsutomu Arakawa Hiroko Tokunaga Matsujiro Ishibashi Masao Tokunaga |
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Institution: | (1) Biochemistry and Applied Biosciences, The United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan;(2) Alliance Protein Laboratory, 3957 Corte Cancion, Thousand Oaks, CA 91360, USA;(3) Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan; |
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Abstract: | Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant
tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity
from Escherichia coli. The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic
stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum
of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting
that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure.
This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw
starch granules and amylose resin with 70–80% efficiency compared with binding of equimolar His-SBD12. Although the thermal
unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different:
His-SBD12 refolded slowly (T1/2 = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T1/2 = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5–6 °C
increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding
were insignificant. |
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