Identification of a novel protein with guanylyl cyclase activity in Arabidopsis thaliana
Identification of a novel protein with guanylyl cyclase activity in Arabidopsis thaliana
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DOI：
10.1074/jbc.m210983200
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发表时间：
2003-02-21
影响因子：
4.8
通讯作者：
Gehring, C
中科院分区：
文献类型：
--
作者：
Ludidi, N;Gehring, C
鸟苷环化酶(GCs)催化5‘-三磷酸鸟苷(GTP)合成第二信使3’，5‘-环磷酸鸟苷(CGMP)。虽然在植物中有许多cGMP介导的过程已被报道，但还没有发现具有GC活性的植物分子。当用蓝藻的GC序列查询拟南芥基因组时，低等和高等真核生物没有发现具有显著相似性的未分配蛋白质。然而，根据注释的GCs催化中心中保守的和功能分配的氨基酸对A.thaliana基因组进行基序搜索，得到一个候选的GCs也包含邻近的富含甘氨酸的结构域。在这种被称为AtGC1的分子中，催化结构域位于N-末端。AtGC1含有参与与鸟氨酸氢键的精氨酸或赖氨酸，以及赋予GTP底物专一性的半胱氨酸。当AtGC1在大肠杆菌中表达时，细胞提取物产生的cGMP是对照提取物的2.5倍，而且这种增加不依赖于一氧化氮。此外，纯化的重组AtGC1在体外具有依赖于镁离子的GC活性，而在没有GTP的情况下，以ATP为底物时，腺酰环化酶的活性降低了3倍。体外催化活性证明，AtGC1既可以作为单体发挥作用，也可以作为齐聚物发挥作用。因此，AtGc1不仅是第一个具有功能的植物GC，而且由于其独特的结构域结构，它也是一类新的GC的成员。
Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) from guanosine 5'-triphosphate (GTP). While many cGMP-mediated processes in plants have been reported, no plant molecule with GC activity has been identified. When the Arabidopsis thaliana genome is queried with GC sequences from cyanobacteria, lower and higher eukaryotes no unassigned proteins with significant similarity are found. However, a motif search of the A. thaliana genome based on conserved and functionally assigned amino acids in the catalytic center of annotated GCs returns one candidate that also contains the adjacent glycine-rich domain typical for GCs. In this molecule, termed AtGC1, the catalytic domain is in the N-terminal part. AtGC1 contains the arginine or lysine that participates in hydrogen bonding with guanine and the cysteine that confers substrate specificity for GTP. When AtGC1 is expressed in Escherichia coli, cell extracts yield >2.5 times more cGMP than control extracts and this increase is not nitric oxide dependent. Furthermore, purified recombinant AtGC1 has Mg2+-dependent GC activity in vitro and >3 times less adenylyl cyclase activity when assayed with ATP as substrate in the absence of GTP. Catalytic activity in vitro proves that AtGC1 can function either as a monomer or homo-oligomer. AtGC1 is thus not only the first functional plant GC but also, due to its unusual domain organization, a member of a new class of GCs.