Master Emma R, Zheng Yun, Storms Reginald, Tsang Adrian, Powlowski Justin
Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, QC, Canada H4B 1R6.
Biochem J. 2008 Apr 1;411(1):161-70. doi: 10.1042/BJ20070819.
A new GH12 (glycosyl hydrolase 12) family XEG [xyloglucan-specific endo-beta-1,4-glucanase (EC 3.2.1.151)] from Aspergillus niger, AnXEG12A, was overexpressed, purified and characterized. Whereas seven xyloglucanases from GH74 and two xyloglucanases from GH5 have been characterized previously, this is only the third characterized example of a GH12 family xyloglucanase. GH12 enzymes are structurally and mechanistically distinct from GH74 enzymes. Although over 100 GH12 sequences are now available, little is known about the structural and biochemical bases of xyloglucan binding and hydrolysis by GH12 enzymes. Comparison of the AnXEG12A cDNA sequence with the genome sequence of A. niger showed the presence of two introns, one in the coding region and the second one in the 333-nt-long 3'-untranslated region of the transcript. The enzyme was expressed recombinantly in A. niger and was readily purified from the culture supernatant. The isolated enzyme appeared to have been processed by a kexin-type protease, which removed a short prosequence. The substrate specificity was restricted to xyloglucan, with cleavage at unbranched glucose in the backbone. The apparent kinetic parameters were similar to those reported for other xyloglucan-degrading endoglucanases. The pH optimum (5.0) and temperature resulting in highest enzyme activity (50-60 degrees C) were higher than those reported for a GH12 family xyloglucanase from Aspergillus aculeatus, but similar to those of cellulose-specific endoglucanases from the GH12 family. Phylogenetic, sequence and structural comparisons of GH12 family endoglucanases helped to delineate features that appear to be correlated to xyloglucan specificity.
对来自黑曲霉的一种新的GH12（糖基水解酶12）家族木葡聚糖特异性内切β-1,4-葡聚糖酶（EC 3.2.1.151）AnXEG12A进行了过表达、纯化及特性鉴定。此前已对7种来自GH74家族的木葡聚糖酶和2种来自GH5家族的木葡聚糖酶进行了特性鉴定，而这是GH12家族木葡聚糖酶中第三个被鉴定特性的实例。GH12酶在结构和作用机制上与GH74酶不同。尽管目前已有100多个GH12序列，但对于GH12酶结合和水解木葡聚糖的结构及生化基础知之甚少。将AnXEG12A的cDNA序列与黑曲霉的基因组序列进行比较，发现存在两个内含子，一个在编码区，另一个在转录本333 nt长的3'非翻译区。该酶在黑曲霉中进行重组表达，并很容易从培养上清液中纯化出来。分离得到的酶似乎经过了一种克新类型蛋白酶的加工，去除了一段短的前导序列。底物特异性仅限于木葡聚糖，在主链中无分支的葡萄糖处进行切割。表观动力学参数与其他降解木葡聚糖的内切葡聚糖酶报道的参数相似。最适pH（5.0）和产生最高酶活性的温度（50 - 60℃）高于来自棘孢曲霉的GH12家族木葡聚糖酶报道的值，但与来自GH12家族的纤维素特异性内切葡聚糖酶相似。GH12家族内切葡聚糖酶的系统发育、序列和结构比较有助于描绘出似乎与木葡聚糖特异性相关的特征。
