Wagner K U, Masepohl B, Pistorius E K
Universität Bielefeld, FRG.
Microbiology (Reading). 1995 Dec;141 ( Pt 12):3049-58. doi: 10.1099/13500872-141-12-3049.
A gene (phoV) encoding an alkaline phosphatase from Synechococcus sp. strain PCC 7942 was isolated by screening a plasmid gene bank for expression of alkaline phosphatase activity in Escherichia coli JM103. Two independent clones carrying the same alkaline-phosphatase-encoding gene were isolated. One of these clones (pKW1) was further analysed and the nucleotide sequence of a contiguous 3234 bp DNA fragment was determined. Two complete open reading frames (ORF1 and phoV) and an incomplete ORF3 were identified reading in the same direction. The deduced phoV gene product showed 34% identity to the alkaline phosphatase PhoA from Zymomonas mobilis, and the N-terminal part of the putative ORF3 protein exhibited 57% identity to a protein of unknown function from Frankia sp. Insertional inactivation of the Synechococcus PCC 7942 phoV gene failed, indicating an essential role for either the phoV or the ORF3 gene product. PhoV consists of 550 amino acid residues, resulting in a molecular mass of 61.3 kDa. To overexpress the Synechococcus PCC 7942 phoV gene in E. coli, plasmid pKW1 was transformed into a phoA mutant of E. coli (CC118). In E. coli strain CC118(pKW1) PhoV was expressed constitutively with high rates of activity, and was shown to be membrane associated in the periplasmic space. After partial purification of the recombinant PhoV, it was shown that, like other alkaline phosphatases, the Synechococcus PhoV had a broad pH optimum in the alkaline region and a broad substrate specificity for phosphomonoesters, required Zn2+ for activity, and was inhibited by phosphate. In contrast to several other alkaline phosphatases, PhoV was inhibited by Mn2+. Due to the lack of a Synechococcus PCC 7942 phoV mutant strain, the function of PhoV remains uncertain. However, the present results show that Synechococcus PCC 7942 has a second, probably phosphate-irrepressible, alkaline phosphatase (PhoV, 61.3 kDa) in addition to the phosphate-repressible enzyme (PhoA, 145 kDa) already described.
通过筛选质粒基因文库以检测碱性磷酸酶活性在大肠杆菌JM103中的表达，分离出了编码来自聚球藻属（Synechococcus sp.）菌株PCC 7942碱性磷酸酶的基因（phoV）。分离出了两个携带相同碱性磷酸酶编码基因的独立克隆。对其中一个克隆（pKW1）进行了进一步分析，并测定了一个连续3234 bp DNA片段的核苷酸序列。鉴定出两个完整的开放阅读框（ORF1和phoV）以及一个不完整的ORF3，它们的阅读方向相同。推导的phoV基因产物与运动发酵单胞菌（Zymomonas mobilis）的碱性磷酸酶PhoA具有34%的同一性，并且推定的ORF3蛋白的N端部分与弗兰克氏菌属（Frankia sp.）一种功能未知的蛋白具有57%的同一性。聚球藻PCC 7942 phoV基因的插入失活未成功，这表明phoV或ORF3基因产物具有重要作用。PhoV由550个氨基酸残基组成，分子量为61.3 kDa。为了在大肠杆菌中过表达聚球藻PCC 7942 phoV基因，将质粒pKW1转化到大肠杆菌（CC118）的一个phoA突变体中。在大肠杆菌菌株CC118(pKW1)中，PhoV组成型表达且活性很高，并且显示与周质空间中的膜相关。对重组PhoV进行部分纯化后发现，与其他碱性磷酸酶一样，聚球藻PhoV在碱性区域具有较宽的最适pH，对磷酸单酯具有较宽的底物特异性，活性需要Zn2+，并且受到磷酸盐的抑制。与其他几种碱性磷酸酶不同，PhoV受到Mn2+的抑制。由于缺乏聚球藻PCC 7942 phoV突变体菌株，PhoV的功能仍然不确定。然而，目前的结果表明，除了已经描述的可被磷酸盐阻遏的酶（PhoA，145 kDa）之外，聚球藻PCC 7942还有第二种可能不可被磷酸盐阻遏的碱性磷酸酶（PhoV，61.3 kDa）。
