Department of Biological Sciences, Idaho State University, Pocatello, ID 83209, USA.
BMC Plant Biol. 2010 Jun 18;10:118. doi: 10.1186/1471-2229-10-118.
Protein prenylation is a common post-translational modification in metazoans, protozoans, fungi, and plants. This modification, which mediates protein-membrane and protein-protein interactions, is characterized by the covalent attachment of a fifteen-carbon farnesyl or twenty-carbon geranylgeranyl group to the cysteine residue of a carboxyl terminal CaaX motif. In Arabidopsis, era1 mutants lacking protein farnesyltransferase exhibit enlarged meristems, supernumerary floral organs, an enhanced response to abscisic acid (ABA), and drought tolerance. In contrast, ggb mutants lacking protein geranylgeranyltransferase type 1 exhibit subtle changes in ABA and auxin responsiveness, but develop normally.
We have expressed recombinant Arabidopsis protein farnesyltransferase (PFT) and protein geranylgeranyltransferase type 1 (PGGT1) in E. coli and characterized purified enzymes with respect to kinetic constants and substrate specificities. Our results indicate that, whereas PFT exhibits little specificity for the terminal amino acid of the CaaX motif, PGGT1 exclusively prenylates CaaX proteins with a leucine in the terminal position. Moreover, we found that different substrates exhibit similar K(m) but different k(cat) values in the presence of PFT and PGGT1, indicating that substrate specificities are determined primarily by reactivity rather than binding affinity.
The data presented here potentially explain the relatively strong phenotype of era1 mutants and weak phenotype of ggb mutants. Specifically, the substrate specificities of PFT and PGGT1 suggest that PFT can compensate for loss of PGGT1 in ggb mutants more effectively than PGGT1 can compensate for loss of PFT in era1 mutants. Moreover, our results indicate that PFT and PGGT1 substrate specificities are primarily due to differences in catalysis, rather than differences in substrate binding.
蛋白异戊烯化是后生动物、原生动物、真菌和植物中常见的翻译后修饰。这种修饰通过将十五碳法呢基或二十碳香叶基共价连接到羧基末端 CaaX 基序的半胱氨酸残基上，介导蛋白质-膜和蛋白质-蛋白质相互作用。在拟南芥中，缺乏蛋白法尼基转移酶的 era1 突变体表现出分生组织增大、花器官增多、对脱落酸（ABA）的反应增强和耐旱性。相比之下，缺乏蛋白香叶基转移酶 1（PGGT1）的 ggb 突变体在 ABA 和生长素反应方面只有细微变化，但发育正常。
我们在大肠杆菌中表达了重组拟南芥蛋白法尼基转移酶（PFT）和蛋白香叶基转移酶 1（PGGT1），并对纯化酶的动力学常数和底物特异性进行了表征。我们的结果表明，尽管 PFT 对 CaaX 基序的末端氨基酸没有特异性，但 PGGT1 仅将末端位置为亮氨酸的 CaaX 蛋白异戊烯化。此外，我们发现不同的底物在 PFT 和 PGGT1 存在的情况下表现出相似的 K(m)但不同的 k(cat)值，表明底物特异性主要由反应性而非结合亲和力决定。
这里提出的数据可能解释了 era1 突变体较强的表型和 ggb 突变体较弱的表型。具体而言，PFT 和 PGGT1 的底物特异性表明，PFT 可以比 PGGT1 更有效地补偿 ggb 突变体中 PGGT1 的缺失，而 PGGT1 不能有效地补偿 era1 突变体中 PFT 的缺失。此外，我们的结果表明，PFT 和 PGGT1 的底物特异性主要是由于催化的差异，而不是由于底物结合的差异。
