Gilbert H J, Hazlewood G P, Laurie J I, Orpin C G, Xue G P
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, UK.
Mol Microbiol. 1992 Aug;6(15):2065-72. doi: 10.1111/j.1365-2958.1992.tb01379.x.
A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.
从瘤胃厌氧真菌梨形新丽鞭毛虫提取的mRNA构建的cDNA文库中分离出一个编码木聚糖酶A（XYLA）的cDNA（xynA）。从携带xynA的大肠杆菌中纯化得到的重组XYLA，其相对分子质量为53,000，可将燕麦-斯佩尔特木聚糖水解为木二糖和木糖。该酶不水解任何纤维素底物。xynA的核苷酸序列显示有一个1821 bp的单一开放阅读框，编码一个相对分子质量为66,192的蛋白质。预测的XYLA一级结构包括一个N端信号肽，其后是一个225个氨基酸的重复序列，该重复序列通过苏氨酸/脯氨酸连接序列与串联的40个残基的C端重复序列分开。大的N端重复区域由不同的催化结构域组成，这些结构域对全长酶表现出相似的底物特异性。XYLA的重复结构表明该酶源自一个经历了两次离散复制的祖先基因。序列比较分析显示XYLA与属于纤维素酶/木聚糖酶家族G的细菌木聚糖酶之间有显著的同源性。这些同源酶之一源自瘤胃细菌黄化瘤胃球菌。XYLA与瘤胃原核生物木聚糖酶之间观察到的同源性可能是瘤胃原核生物和低等真核生物之间基因水平转移的结果，无论是在这些生物栖息于瘤胃时，还是在它们定殖于反刍动物之前。还应注意的是，梨形新丽鞭毛虫XYLA是由重复序列组成的木聚糖酶的第一个例子。其他瘤胃真菌植物细胞壁水解酶是否也存在这种普遍现象还有待确定。
