Voynova Natalia E, Fu Zhuji, Battaile Kevin P, Herdendorf Timothy J, Kim Jung-Ja P, Miziorko Henry M
Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, MO 64110, USA.
Arch Biochem Biophys. 2008 Dec 1;480(1):58-67. doi: 10.1016/j.abb.2008.08.024. Epub 2008 Sep 18.
Expression in Escherichia 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 (2.4A resolution). hMDD exhibits V(max)=6.1+/-0.5 U/mg; K(m) for ATP is 0.69+/-0.07 mM and K(m) for (R,S) mevalonate diphosphate is 28.9+/-3.3 microM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a approximately 1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, comparably to wild-type enzyme. Diphosphoglycolyl proline (K(i)=2.3+/-0.3 uM) and 6-fluoromevalonate 5-diphosphate (K(i)=62+/-5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a V(max)=0.25+/-0.0 2U/mg and a 15-fold inflation in K(m) for mevalonate diphosphate. N17A's K(i) 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 (2.8A) 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.
带有组氨酸标签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以及晶体结构测定（分辨率为2.4埃）。hMDD的V(max)=6.1±0.5 U/mg；ATP的K(m)为0.69±0.07 mM，(R,S)甲羟戊酸二磷酸的K(m)为28.9±3.3 μM。预测位于hMDD活性位点的保守极性残基被突变以测试其功能重要性。R161Q的比活性降低了约1000倍，而与野生型酶相比，其与荧光底物类似物TNP-ATP的结合能力相当。二磷酸乙醇酰脯氨酸（K(i)=2.3±0.3 μM）和6-氟甲羟戊酸5-二磷酸（K(i)=62±5 nM）是甲羟戊酸二磷酸的竞争性抑制剂。N17A的V(max)=0.25±0.02 U/mg，甲羟戊酸二磷酸的K(m)增加了15倍。与野生型酶相比，N17A对二磷酸乙醇酰脯氨酸和氟甲羟戊酸二磷酸的K(i)值增加（分别>70倍和40倍）。hMDD结构表明位于活性位点裂隙内部口袋中的R161和N17之间的距离较近（2.8埃）。数据表明R161和N17在甲羟戊酸二磷酸的结合和定向中具有功能重要性。
