Residue Phe112 of the Human-Type Corrinoid Adenosyltransferase (PduO) Enzyme of Lactobacillus reuteri Is Critical to the Formation of the Four-Coordinate Co(II) Corrinoid Substrate and to the Activity of the Enzyme
Residue Phe112 of the Human-Type Corrinoid Adenosyltransferase (PduO) Enzyme of Lactobacillus reuteri Is Critical to the Formation of the Four-Coordinate Co(II) Corrinoid Substrate and to the Activity of the Enzyme
复制标题
DOI：
10.1021/bi9000134
复制
发表时间：
2009-04-14
期刊：
影响因子：
2.9
通讯作者：
Escalante-Semerena, Jorge C.
中科院分区：
文献类型：
--
作者：
Mera, Paola E.;St Maurice, Martin;Escalante-Semerena, Jorge C.
ATP：类柯啉腺苷转移酶（ACAS）催化腺苷部分从ATP转移到cob（I）alamin，通过四配位cob（II）alamin中间体。目前，还不清楚ACA如何促进活性所需的四配位类咕啉物质的形成。已发表的来自罗伊氏乳杆菌（LrPduO）的ACA与ATP和cob（II）维生素复合的高分辨率晶体结构显示咕啉环的a面周围的环境由庞大的疏水残基组成。为了了解这些残基是如何促进四配位辅酶（II）丙氨酸生成的，从L。reuteri（LrPduO）的动力学和结构特征。这些研究表明，残基Phe 112在取代5，6-二甲基苯并咪唑（DMB）与环上的Co离子的配位键中是关键的，导致形成四配位物种。F112 A取代导致酶的催化效率下降80%。这种活性损失的解释是从突变蛋白的晶体结构中获得的，其显示cob（II）alamin结合在活性位点中，DMB与钴离子配位。LrPduO（F112 H）变体的晶体结构显示了类可啉和酶之间的DMB-off/His-on相互作用，其催化效率比野生型蛋白低4个数量级。对LrPduO（F112 H）的动力学参数的分析表明，F112 H取代对产物释放产生负面影响。在Cbl结合口袋中的其他疏水残基的取代没有导致体外催化效率的显著缺陷;然而，在这项工作中分析的变体酶都不支持Cbl体内生物合成。
ATP:Corrinoid adenosyltransferases (ACAS) catalyze the transfer of the adenosyl moiety from ATP to cob(I)alamin via a four-coordinate cob(II)alamin intermediate. At present, it is unknown how ACAs promote the formation of the four-coordinate corrinoid species needed for activity. The published high-resolution crystal structure of the ACA from Lactobacillus reuteri (LrPduO) in complex with ATP and cob(II)alamin shows that the environment around the a face of the corrin ring consists of bulky hydrophobic residues. To understand how these residues promote the generation of the four-coordinate cob(II)alamin, variants of the human-type ACA enzyme from L. reuteri (LrPduO) were kinetically and structurally characterized. These studies revealed that residue Phe112 is critical in the displacement of 5,6-dimethylbenzimidazole (DMB) from its coordination bond with the Co ion of the ring, resulting in the formation of the four-coordinate species. An F112A substitution resulted in a 80% drop in the catalytic efficiency of the enzyme. The explanation for this loss of activity was obtained from the crystal structure of the mutant protein, which showed cob(II)alamin bound in the active site with DMB coordinated to the cobalt ion. The crystal structure of an LrPduO(F112H) variant showed a DMB-off/His-on interaction between the corrinoid and the enzyme, whose catalytic efficiency was 4 orders of magnitude lower than that of the wild-type protein. The analysis of the kinetic parameters of LrPduO(F112H) suggests that the F112H substitution negatively impacts product release. Substitutions of other hydrophobic residues in the Cbl binding pocket did not result in significant defects in catalytic efficiency in vitro; however, none of the variant enzymes analyzed in this work supported AdoCbl biosynthesis in vivo.