Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure.
Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure.
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DOI：
10.1016/j.abb.2008.08.024
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发表时间：
2008-12-01
影响因子：
3.9
通讯作者：
Miziorko HM
中科院分区：
文献类型：
--
作者：
Voynova NE;Fu Z;Battaile KP;Herdendorf TJ;Kim JJ;Miziorko HM
他标记的人甲羟戊酸二磷酸脱羧酶（hMDD）在大肠杆菌中的表达加快了酶的分离、表征、甲羟戊酸二磷酸结合位点的功能研究和晶体结构测定（2.4 Å分辨率）。hMDD Vmax = 6.1±0.5 U/mg；ATP的Km为0.69±0.07 mM, （R，S）甲羟戊酸二磷酸的Km为28.9±3.3 uM。预测在hMDD活性位点的保守极性残基被突变以测试功能的重要性。R161Q表现出约1000倍的比活性降低，而与荧光底物类似物TNP-ATP结合，如野生型酶。二磷酸甘酰脯氨酸（Ki = 2.3±0.3 uM）和6-氟戊酸5-二磷酸（Ki = 62±5 nM）是相对于二磷酸甲戊酸的竞争性抑制剂。N17A对二磷酸甲戊酸盐的Vmax = 0.25±0.02 U/mg，在Km内膨胀15倍。与野生型酶相比，N17A对二磷酸甘酰脯氨酸和氟戊酸二磷酸的Ki值升高（分别为70倍和40倍）。hMDD结构表明R161和N17之间的距离（2.8 Å），它们位于活性位点间隙的内部口袋中。这些数据表明R161和N17在甲羟戊酸二磷酸的结合和取向中具有重要的功能。
Expression in E. coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4 Å resolution). hMDD exhibits Vmax = 6.1 ± 0.5 U/mg; Km for ATP is 0.69 ± 0.07 mM and Km for (R,S) mevalonate diphosphate is 28.9 ± 3.3 uM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a ~1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, like wild-type enzyme. Diphosphoglycolyl proline (Ki = 2.3 ± 0.3 uM) and 6-fluoromevalonate 5-diphosphate (Ki = 62 ± 5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a Vmax = 0.25 ± 0.02 U/mg and a 15-fold inflation in Km for mevalonate diphosphate. N17A’s Ki values for diphosphoglycolyl proline and fluoromevalonate diphosphate are inflated (>70-fold and 40-fold, respectively) in comparison with wild-type enzyme. hMDD structure indicates the proximity (2.8 Å) between R161 and N17, which are located in an interior pocket of the active site cleft. The data suggest the functional importance of R161 and N17 in the binding and orientation of mevalonate diphosphate.
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影响因子：
4.8
作者：
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通讯作者：
Kim, JJP
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