Huang Y, Garrison P N, Barnes L D
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284-7760, USA.
Biochem J. 1995 Dec 15;312 ( Pt 3)(Pt 3):925-32. doi: 10.1042/bj3120925.
Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Peña and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Séraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.
二腺苷5',5''-P1,P4-四磷酸（Ap4A）不对称水解酶（EC 3.6.1.17）是粟酒裂殖酵母中发现的一种特异性Ap4A分解代谢酶。我们之前已描述了从粟酒裂殖酵母中部分纯化Ap4A水解酶的过程[罗宾逊、德拉佩尼亚和巴恩斯（1993年），《生物化学与生物物理学报》1161卷，第139 - 148页]。我们测定了Ap4A水解酶N端20个氨基酸的序列，并根据该序列设计了两条简并PCR引物。通过PCR获得的60 bp DNA片段对Ap4A水解酶具有特异性，利用该片段通过筛选多拷贝质粒中的基因组DNA文库从粟酒裂殖酵母中分离出Ap4A水解酶基因aph1。粟酒裂殖酵母阳性转化体粗提上清液中的Ap4A水解酶活性比对照高约25倍。未检测到磷酸盐对酶活性的刺激作用。粟酒裂殖酵母的aph1基因含有三个内含子。通过对粟酒裂殖酵母cDNA文库中aph1基因的cDNA进行测序，确认了内含子边界。aph1基因推导的开放阅读框编码182个氨基酸。在Ap4A水解酶与组氨酸三联体（HIT）蛋白家族[Séraphin（1992年），《DNA序列》3卷，第177 - 179页]之间鉴定出两个显著的局部相似区域。HIT蛋白存在于原核生物、酵母、植物和哺乳动物中。除了牛蛋白在体外抑制蛋白激酶C外，它们的功能尚不清楚。在包括假定的HxHxH锌（2 +）结合基序在内的HIT蛋白中保守的所有四个组氨酸残基在Ap4A水解酶中也保守。此外，酿酒酵母的Ap4A磷酸化酶I和II与粟酒裂殖酵母的Ap4A水解酶之间存在两个相似区域。这些区域与HIT蛋白相似区域重叠。粟酒裂殖酵母的aph1基因是第一个被克隆和测序的不对称Ap4A水解酶基因。
