Arz H E, Gisselmann G, Schiffmann S, Schwenn J D
Biochemistry of Plants, Faculty of Biology, Ruhr-University-Bochum, Germany.
Biochim Biophys Acta. 1994 Aug 2;1218(3):447-52. doi: 10.1016/0167-4781(94)90203-8.
A cDNA clone with an open reading frame of 831 nucleotides was isolated from a lambda ZapII-library of Arabidopsis thaliana. The nucleotide sequence of the cDNA is homologous to the APS-kinase genes from enterobacteria, diazotrophic bacteria, and yeast: Escherichia coli (cys C: 53.2%), Rhizobium meliloti (nod Q: 52.6%), and Saccharomyces cerevisiae (met 14:57.1%). The polypeptide deduced from the plant APS-kinase cDNA is comprised of 276 amino acid residues with a molecular weight of 29,790. It contains an N-terminal extension of 77 amino acids. This extension includes a putative transit peptide of 37 residues separated from the core protein by a VRACV processing site for stromal peptidase; a molecular weight of 26,050 is predicted for the processed protein. The relatedness between bacterial, fungal and plant APS-kinase polypeptides ranges from 47.5% (E. coli), 55.4% (S. cerevisiae), 52.6% (R. meliloti), and 50.3% (Azospirillum brasilense). The plant polypeptide contains eight cysteine residues; two cysteines flank a conserved purine nucleotide binding domain: GxxxxGK. Also conserved are a serine-182 as a possible phosphate transferring group and a K/LARAGxxxxFTG motif described for PAPS dependent enzymes. The identity of the gene was confirmed by analyzing the function of the gene product. The putative transit peptide was deleted by PCR and the truncated gene was expressed in a pTac1 vector system. A polypeptide of MW 25761 could be induced by IPTG. The gene product was enzymatically active as APS-kinase. It produced PAPS from APS and ATP--the absence of ATP but supplemented with thiols, the APS-kinase reacted as APS-sulphotransferase. APS-sulphotransferase is not a separate enzyme but identical with APS-kinase.
从拟南芥的λZapII文库中分离出一个开放阅读框为831个核苷酸的cDNA克隆。该cDNA的核苷酸序列与来自肠杆菌、固氮菌和酵母的APS激酶基因同源：大肠杆菌（cys C：53.2%）、苜蓿根瘤菌（nod Q：52.6%）和酿酒酵母（met 14：57.1%）。从植物APS激酶cDNA推导的多肽由276个氨基酸残基组成，分子量为29,790。它包含一个77个氨基酸的N端延伸。该延伸包括一个37个残基的假定转运肽，通过基质肽酶的VRACV加工位点与核心蛋白分开；预测加工后的蛋白分子量为26,050。细菌、真菌和植物APS激酶多肽之间的相关性范围为47.5%（大肠杆菌）、55.4%（酿酒酵母）、52.6%（苜蓿根瘤菌）和50.3%（巴西固氮螺菌）。植物多肽包含八个半胱氨酸残基；两个半胱氨酸位于保守的嘌呤核苷酸结合域两侧：GxxxxGK。同样保守的是作为可能的磷酸转移基团的丝氨酸-182以及为PAPS依赖性酶描述的K/LARAGxxxxFTG基序。通过分析基因产物的功能证实了该基因的身份。通过PCR删除假定的转运肽，并在pTac1载体系统中表达截短的基因。IPTG可诱导出分子量为25761的多肽。该基因产物具有APS激酶的酶活性。它从APS和ATP产生PAPS——在没有ATP但补充有硫醇的情况下，APS激酶作为APS磺基转移酶起作用。APS磺基转移酶不是一种单独的酶，而是与APS激酶相同。
