Lin Fu-Pang, Chuang Hsu-Han, Liu Yi-Hsuan, Hsieh Chia-Yu, Lin Pei-Wen, Lin Hsu-Yang
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
Arch Microbiol. 2009 Mar;191(3):265-73. doi: 10.1007/s00203-008-0451-x. Epub 2008 Dec 17.
C-terminal truncation mutagenesis was used to explore the functional and structural significance of the C-terminal region of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606) included initial rate kinetics, fluorescence and circular dichroism (CD) spectrometric properties, and substrate binding and hydrolysis abilities. The overall catalytic efficiency, k(cat)/K(M), of AcD1ChiAK606 with the 4MU-(GlcNAc)(2) and the 4MU-(GlcNAc)(3) chitin substrates was 15-26% decreased. When compared with AcD1ChiA, the truncated mutant AcD1ChiAK606 maintained 80% relative substrate-binding ability and about 76% of the hydrolyzing efficiency against the insoluble alpha-chitin substrate. Both fluorescence and CD spectroscopy indicated that AcD1ChiAK606 retained the same conformation as AcD1ChiA. These results indicated that removal of the C-terminal 259 amino acid residues, including the putative chitin-binding motif and the A region (a motif of unknown function) of AcD1ChiA, did not seriously affect the enzyme structure integrity as well as activity. The present study provided evidences illustrating that the binding and hydrolyzing of insoluble chitin substrates by AcD1ChiA were not absolutely dependent on the putative C-terminal chitin-binding domain and the function-unknown A region.
采用C末端截短诱变技术来探究豚鼠气单胞菌D1几丁质酶（AcD1ChiA）C末端区域的功能和结构意义。对工程化全长AcD1ChiA和截短突变体（AcD1ChiAK606）进行的比较研究包括初始速率动力学、荧光和圆二色（CD）光谱特性以及底物结合和水解能力。AcD1ChiAK606对4MU-(GlcNAc)₂和4MU-(GlcNAc)₃几丁质底物的总体催化效率k(cat)/K(M)降低了15 - 26%。与AcD1ChiA相比，截短突变体AcD1ChiAK606对不溶性α-几丁质底物保持80%的相对底物结合能力和约76%的水解效率。荧光光谱和CD光谱均表明AcD1ChiAK606保留了与AcD1ChiA相同的构象。这些结果表明，去除AcD1ChiA的C末端259个氨基酸残基，包括假定的几丁质结合基序和A区域（功能未知的基序），并未严重影响酶的结构完整性和活性。本研究提供的证据表明，AcD1ChiA对不溶性几丁质底物的结合和水解并不绝对依赖于假定的C末端几丁质结合结构域和功能未知的A区域。
