Albert A, Yenush L, Gil-Mascarell M R, Rodriguez P L, Patel S, Martínez-Ripoll M, Blundell T L, Serrano R
Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Serrano 119, Madrid, E-28006, Spain.
J Mol Biol. 2000 Jan 28;295(4):927-38. doi: 10.1006/jmbi.1999.3408.
The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops. We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution. A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity. Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity. Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase. The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity.
酵母HAL2基因的产物（Hal2p）是钠和锂毒性在体内的作用靶点，其过表达可提高酵母和植物的耐盐性。Hal2p是一种代谢磷酸酶，催化3'-磷酸腺苷-5'-磷酸（PAP）水解为AMP。它是PAP磷酸酶进化保守家族的原型，对该组钠不敏感酶进行工程改造可能有助于培育耐盐作物。我们已解析了Hal2p与镁、锂以及PAP水解的两种产物AMP和Pi形成复合物的晶体结构，分辨率为1.6埃。在生长的酵母中对HAL2基因的随机突变进行功能筛选，产生了阳离子敏感性降低的酶形式。对这些突变体的分析确定了一个盐桥（Glu238……Arg152）和一个疏水键（Va170……Trp293）是决定阳离子敏感性的重要框架相互作用。Hal2p属于一个更大的锂敏感磷酸酶超家族，其中包括肌醇单磷酸酶。在Hal2p中发生突变的疏水相互作用在这个超家族中是保守的，其在人肌醇单磷酸酶中的破坏也导致阳离子敏感性降低。
