EVIDENCE FOR A GENERAL ROLE FOR NONCATALYTIC THERMOSTABILIZING DOMAINS IN XYLANASES FROM THERMOPHILIC BACTERIA
EVIDENCE FOR A GENERAL ROLE FOR NONCATALYTIC THERMOSTABILIZING DOMAINS IN XYLANASES FROM THERMOPHILIC BACTERIA
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DOI：
10.1042/bj3070151
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发表时间：
1995-04-01
影响因子：
4.1
通讯作者：
GILBERT, HJ
中科院分区：
文献类型：
--
作者：
FONTES, CMGA;HAZLEWOOD, GP;GILBERT, HJ
在λ ZAP II中构建的热纤梭菌DNA的基因组文库中筛选木聚糖酶表达克隆。交叉杂交实验表明，从基因文库中分离到一个新的木聚糖酶基因，命名为xynY。所编码的酶木聚糖酶Y（XYLY）显示出内切β 1，4-木聚糖酶的特征：该酶快速水解燕麦斯佩耳特燕麦、小麦和黑麦阿拉伯聚糖，并且对甲基伞形酮基-β-D-纤维二糖苷有活性，但不水解任何纤维素底物。该酶的最适pH和最适温度分别为6.8和75 ℃，大肠杆菌表达的重组XYLY的最大M（r）为116000。xynY的核苷酸序列包含一个3228 bp的开放阅读框，编码预测的M（r）120 - 105的蛋白质。编码的酶含有一个典型的N-末端26个残基的信号肽，其次是一个164个氨基酸的序列，指定的结构域A，这是不是必需的催化活性。结构域A的下游是351个残基的木聚糖酶F家族催化结构域，随后是与解糖嗜热厌氧杆菌木聚糖酶A的热稳定结构域具有28%序列同一性的180个残基序列。XYLY的C-末端部分包含在所有其他C. Thermocellum植物细胞壁水解酶是细菌的多酶复合物的组成部分，称为纤维素酶体，随后是286个残基的结构域，其与C.热纤酶Z.该酶不含其他C.热纤植物细胞壁水解酶。分析截短形式的XYLY和杂合蛋白，所述杂合蛋白包含XYLY融合至E. coli麦芽糖结合结构域，证实XYLY含有一个中心催化结构域和一个相邻的热稳定结构域。C-末端结构域不与纤维素或木聚糖结合。使用抗血清对XYLY的蛋白质印迹分析表明，木聚糖酶位于纤维素酶体，并没有出现广泛的糖基化。XYLY的非催化结构域的一般稳定性的嗜热木聚糖酶进行了讨论。
A genomic library of Clostridium thermocellum DNA constructed in lambda ZAPII was screened for xylanase-expressing clones. Cross-hybridization experiments revealed a new xylanase gene isolated from the gene library, which was designated xynY. The encoded enzyme, xylanase Y (XYLY), displayed features characteristic of an endo-beta 1,4-xylanase: the enzyme rapidly hydrolysed oat spelt, wheat and rye arabinoxylans and was active against methyl-umbelliferyl-beta-D-cellobioside, but did not hydrolyse any cellulosic substrates. The pH and temperature optima of the enzyme were 6.8 and 75 degrees C respectively, and the recombinant XYLY, expressed by Escherichia coli had a maximum M(r) of 116 000. The nucleotide sequence of xynY contained an open reading frame of 3228 bp encoding a protein of predicted M(r) 120 105. The encoded enzyme contained a typical N-terminal 26-residue signal peptide, followed by a 164 amino acid sequence, designated domain A, that was not essential for catalytic activity. Downstream of domain A was a 351-residue xylanase Family F catalytic domain, followed by a 180-residue sequence that exhibited 28 % sequence identity with a thermostable domain of Thermoanaerobacterium saccharolyticum xylanase A. The C-terminal portion of XYLY comprised the U-residue duplicated docking sequence found in all other C. thermocellum plant cell wall hydrolases that are constituents of the bacterium's multienzyme complex, termed the cellulosome, followed by a 286-residue domain which exhibited 32 % sequence identity with the N-terminal region of C. thermocellum xylanase Z. The enzyme did not contain linker sequences found in other C. thermocellum plant cell wall hydrolases. Analysis of truncated forms of XYLY and hybrid proteins, comprising segments of XYLY fused to the E. coli maltose binding domain, confirmed that XYLY contained a central catalytic domain and an adjacent thermostable domain. The C-terminal domain did not bind to cellulose or xylan. Western blot analysis using antiserum raised against XYLY showed that the xylanase was located in the cellulosome and did not appear to be extensively glycosylated. The non-catalytic domains of XYLY are discussed in relation to the general stability of thermophilic xylanases.