Mutational and Functional Analyses of Substrate Binding and Catalysis of the Listeria monocytogenes EutT ATP:Co(I)rrinoid Adenosyltransferase
Mutational and Functional Analyses of Substrate Binding and Catalysis of the Listeria monocytogenes EutT ATP:Co(I)rrinoid Adenosyltransferase
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DOI：
10.1021/acs.biochem.0c00078
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发表时间：
2020-03-17
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：
Costa, Flavia G.;Greenhalgh, Elizabeth D.;Escalante-Semerena, Jorge C.
三磷酸腺苷：CO(I)类腺糖基转移酶(ACAT)催化腺苷部分从共底物三磷酸腺苷转移到皮质醇底物。ACAT分为三个系列，即COBA、PduO和EutT。根据活性是否需要二价金属离子，EutT家族酶进一步分为两类(第一类和第二类)。到目前为止，EutT家族中的两类ACAT的结构还没有被阐明。在这项工作中，生物信息学分析结果揭示了EutT同源物的C末端与结构特征的reuri乳杆菌PduO(LrPduO)同源物之间的几个保守残基。在LrPduO中，这些残基与ATP结合和亚基间盐桥的形成有关。这些残基被取代，体内和体外数据支持这样的结论，即无金属(即II类)单核细胞增生性李斯特菌EutT(LmEutT)酶中的等量残基影响ATP结合。在体内和体外分析带有苯丙氨酸和色氨酸残基取代的LmEutT变异体的结果表明，第72位苯丙氨酸残基的取代影响底物结合部位的进入，而第238位色氨酸残基的取代影响Cbl底物与活性部位的结合。与PduO家族的ACAT不同，单个苯丙氨酸残基不是a-配体移位的原因。综上所述，这些数据表明，虽然EutT酶与PduO家族ACAT有一个保守的ATP结合基序和一个亚基间盐桥，但II类EutT家族ACAT利用一种不同于PduO和CobA家族ACAT的未知机制来进行Cb1低配体的置换和还原。
ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely, CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (class I and class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP binding and formation of an intersubunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affected access to the substrate-binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the a-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an intersubunit salt bridge with PduO family ACATs, class II EutT family ACATs utilize an unidentified mechanism for Cbl lower-ligand displacement and reduction that is different from that of PduO and CobA family ACATs.