Lunn J E, Price G D, Furbank R T
CSIRO Plant Industry, Canberra, ACT, Australia.
Plant Mol Biol. 1999 May;40(2):297-305. doi: 10.1023/a:1006130802706.
Sucrose is one of several low-molecular-weight compounds that cyanobacteria accumulate in response to osmotic stress and which are believed to act as osmoprotectants. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains a 2163 bp open reading frame (ORF) that shows similarity to genes from higher plants encoding sucrose-phosphate synthase (SPS), the enzyme responsible for sucrose synthesis. The deduced amino acid sequence shows 35-39% identity with known higher-plant SPS sequences. The putative Synechocystis sps gene was cloned from genomic DNA by PCR amplification and expressed as a His6-tagged amino-terminal fusion protein in Escherichia coli. The expressed protein was purified and shown to be a functional SPS enzyme, confirming the identity of the ORF, which is the first sps gene to be cloned from a prokaryotic organism. The Synechocystis SPS has a molecular mass of 81.5 kDa, which is smaller than the typical higher-plant SPS subunit (117-119 kDa), and lacks the phosphorylation site motifs associated with light- and osmotic stress-induced regulation of SPS in higher plants. The enzyme has Km values for UDPG1c and Fru6P of 2.9 mM and 0.22 mM, respectively, with a Vmax of 17 micromol per minute per mg protein and a pH optimum of 8.5. Unlike the higher-plant enzyme, ADPG1c, CDPG1c and GDPG1c can substitute for UDPG1c as the glucosyl donor with Km values of 2.5, 7.2 and 1.8 mM, respectively. The enzyme is activated by Mg2+ but not by G1c6P, and is only weakly inhibited by inorganic phosphate. The purified protein was used to raise a high-titre antiserum, which recognises a low-abundance 81 kDa protein in Synechocystis sp. PCC 6803 extracts. There was no apparent increase in expression of the 81 kDa protein when the cells were exposed to moderate salt stress, and SPS activity was very low in extracts from both unstressed and salt-stressed cells. These results and the lack of evidence for sucrose accumulation in Synechocystis sp. PCC6803 lead to the conclusion that expression of the sps gene plays no obvious role in adaptation to osmotic stress in this species.
蔗糖是蓝细菌在渗透胁迫下积累的几种低分子量化合物之一，被认为可作为渗透保护剂。集胞藻6803（Synechocystis sp. PCC 6803）的基因组包含一个2163 bp的开放阅读框（ORF），该阅读框与高等植物中编码蔗糖磷酸合酶（SPS）的基因相似，SPS是负责蔗糖合成的酶。推导的氨基酸序列与已知的高等植物SPS序列具有35 - 39%的同一性。通过PCR扩增从基因组DNA中克隆了集胞藻假定的sps基因，并在大肠杆菌中作为His6标签的氨基末端融合蛋白表达。表达的蛋白被纯化并显示为一种功能性SPS酶，证实了该ORF的身份，这是从原核生物中克隆的第一个sps基因。集胞藻SPS的分子量为81.5 kDa，比典型的高等植物SPS亚基（117 - 119 kDa）小，并且缺乏与高等植物中光和渗透胁迫诱导的SPS调节相关的磷酸化位点基序。该酶对UDPG1c和Fru6P的Km值分别为2.9 mM和0.22 mM，Vmax为每毫克蛋白每分钟17微摩尔，最适pH为8.5。与高等植物的酶不同，ADPG1c、CDPG1c和GDPG1c可以替代UDPG1c作为葡萄糖基供体，Km值分别为2.5、7.2和1.8 mM。该酶被Mg2+激活，但不被G1c6P激活，仅被无机磷酸微弱抑制。纯化的蛋白用于制备高效价抗血清，该抗血清可识别集胞藻PCC 6803提取物中一种低丰度的81 kDa蛋白。当细胞暴露于中度盐胁迫时，81 kDa蛋白的表达没有明显增加，并且在未胁迫和盐胁迫细胞的提取物中SPS活性都非常低。这些结果以及在集胞藻PCC6803中缺乏蔗糖积累的证据导致这样的结论：sps基因的表达在该物种适应渗透胁迫中没有明显作用。
