Mechanistic diversity in the RuBisCO superfamily:: The "Enolase" in the methionine salvage pathway in Geobacillus kaustophilus
Mechanistic diversity in the RuBisCO superfamily:: The "Enolase" in the methionine salvage pathway in Geobacillus kaustophilus
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DOI：
10.1021/bi7000483
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发表时间：
2007-04-03
期刊：
影响因子：
2.9
通讯作者：
Gerlt, John A.
中科院分区：
文献类型：
--
作者：
Imker, Heidi J.;Fedorov, Alexander A.;Gerlt, John A.
D-核酮糖1，5-二磷酸羧化酶/加氧酶（RuBisCO）是最丰富的酶，是最近公认的机制多样的RuBisCO超家族的范例成员。RuBisCO反应是通过羧化Lys 201（菠菜酶）的氨基甲酸酯氧从D-核酮糖1，5-二磷酸底物的C3中提取质子而引发的。在杆菌属物种中发现的RuBisCO的异功能同源物在甲硫氨酸补救途径中催化2，3-二酮-5-甲硫基戊烷1-磷酸（DK-MTP 1-P）的互变异构化（“烯醇化”），其中衍生自5 '-甲硫基腺苷的5-甲硫基-D-核糖（MTR）转化为甲硫氨酸[Ashida，H.，Saito，Y.，Kojima，C.，小林，K.，小笠原，N.，和Yokota，A.（2003）A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO，Science 302，286-290]。由该“烯醇化酶”催化的反应通过从DK-MTP 1-P底物的C1提取质子以形成互变异构化产物（共辄烯醇）来完成。由于RuBisCO-和“烯醇化酶”催化的反应在质子提取的区域化学上不同，但预期通过与活性位点Mg 2+配位来共享烯醇化阴离子中间体的稳定，因此我们试图建立“烯醇化酶”反应的结构-功能关系，以便可以建立功能多样性的结构基础。我们确定了由枯草芽孢杆菌和嗜热土芽孢杆菌的“烯醇酶”催化的反应的立体化学过程。使用衍生自D-核糖的替代底物（MTR中的5-OH基团代替5-甲硫基）以及天然DK-MTP 1-P底物的立体特异性氘代样品，我们确定“烯醇化酶”催化的反应涉及1-proS质子的提取。我们还测定了G.嗜碱菌（在Lys 173上羧基化）与Mg 2+和2，3-二酮己烷1-磷酸（稳定的替代底物）配体。质子提取的立体特异性将一般碱基的位置限制在N-末端α + β结构域，而不是C-末端（β/α）（8）-含有羧基化赖氨酸173的桶结构域。在所有“烯醇化酶”中保守的N-末端结构域中的Lys 98定位为提取1-proS质子。与该功能一致，G.嗜碱菌“烯醇化酶”不能催化“烯醇化酶”反应。因此，我们得出结论，RuBisCO超家族的这个功能上不同的成员使用与RuBisCO相同的结构策略来稳定烯醇盐阴离子中间体，即，配位到必需的Mg 2+，但质子提取由不同的一般碱催化。
D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the D- ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ("enolization") of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-D-ribose (MTR) derived from 5'-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this "enolase" is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO- and "enolase"-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg2+, we sought to establish structure-function relationships for the "enolase" reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the "enolases" from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from D-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the "enolase"-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated "enolase" from G. kaustophilus (carboxylated on Lys 173) liganded with Mg2+ and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal alpha+beta domain instead of the C-terminal (beta/alpha)(8)-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all "enolases", is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus "enolase" is unable to catalyze the "enolase" reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base.