Sawai-Hatanaka H, Ashikari T, Tanaka Y, Asada Y, Nakayama T, Minakata H, Kunishima N, Fukuyama K, Yamada H, Shibano Y
Suntory Research Center, Suntory Ltd., Osaka, Japan.
Biosci Biotechnol Biochem. 1995 Jul;59(7):1221-8. doi: 10.1271/bbb.59.1221.
To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, and the evolutionary relationship of the A.ramosus peroxidase (ARP) with the other peroxidases, we isolated complementary and genomic DNA clones encoding ARP and characterized them. The sequence analyses of the ARP and cDNA coding for ARP showed that a mature ARP consists of 344 amino acids with a N-terminal pyroglutamic acid preceded by a signal peptide of 20 amino acid residues. The amino acid sequence of ARP was 99% identical to that of the peroxidase of Coprinus cinereus, a basidiomycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lignin peroxidase activities for ARP when assayed with lignin model compounds. We could identified His184 and His56 as proximal and distal ligands to heme, respectively, and Arg52 as an essential Arg. Comparison of the sequences of complementary and genomic DNAs found that protein-encoding DNA is interrupted by 14 intervening sequences. The ARP cDNA was expressed in the yeast Saccharomyces cerevisiae under the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 units/ml of a secreted active peroxidase.
为了了解丝状子囊菌新分类群——枝状节丛孢过氧化物酶的结构与功能之间的关系，以及枝状节丛孢过氧化物酶（ARP）与其他过氧化物酶的进化关系，我们分离了编码ARP的互补DNA和基因组DNA克隆并对其进行了表征。ARP及其编码cDNA的序列分析表明，成熟的ARP由344个氨基酸组成，N端为焦谷氨酸，前面有一个20个氨基酸残基的信号肽。ARP的氨基酸序列与担子菌灰盖鬼伞过氧化物酶的氨基酸序列99%相同，并且与担子菌木质素过氧化物酶的氨基酸序列也有非常高的相似性（同一性为41 - 43%），尽管在用木质素模型化合物检测时我们未发现ARP具有木质素过氧化物酶活性。我们可以分别鉴定出His184和His56作为血红素的近端和远端配体，以及Arg52作为必需的精氨酸。互补DNA和基因组DNA序列的比较发现，编码蛋白质的DNA被14个间隔序列打断。ARP cDNA在甘油醛 - 3 - 磷酸脱氢酶基因启动子的驱动下在酿酒酵母中表达，产生了0.02单位/毫升的分泌型活性过氧化物酶。
