Characterization of auxiliary iron-sulfur clusters in a radical S-adenosylmethionine enzyme PqqE from Methylobacterium extorquens AM1.
Characterization of auxiliary iron-sulfur clusters in a radical S-adenosylmethionine enzyme PqqE from Methylobacterium extorquens AM1.
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DOI：
10.1002/2211-5463.12314
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发表时间：
2017-12
期刊：
影响因子：
2.6
通讯作者：
Frébortová J
中科院分区：
文献类型：
--
作者：
Saichana N;Tanizawa K;Ueno H;Pechoušek J;Novák P;Frébortová J
PqqE是一种可催化吡咯喹啉醌（PQQ）生物合成初始反应的S -腺苷基- 1 -蛋氨酸（SAM）酶。PqqE属于自由基SAM超家族的SPASM （subtilosin/PQQ/厌氧硫酸酯酶/分枝杆菌素成熟酶）亚家族，包含多个Fe-S簇。为了鉴定来自Methylobacterium extorquens AM1的PqqE中的Fe-S簇，在N端特征基序（CX 3 CX 2C）和C端7 -半胱氨酸基序（CX 9-15 GX 4 CX N CX 2 CX 5 CX 3 CX nC; N =一个未指定的数字）中保守的Cys残基被单独或同时突变为Ser。纯化和重组的突变酶的生化和Mössbauer光谱分析证实了PqqE中存在三个Fe-S簇：N端区域的一个[4Fe-4S]2+簇是SAM还原均溶裂解为甲硫氨酸和5 ' -脱氧腺苷自由基所必需的，C端SPASM区域的[4Fe-4S]2+和[2Fe-2S]2+辅助簇，被认为是在隐藏的活性位点和蛋白质表面之间进行电子转移。[2Fe-2S]2+簇的存在是自由基SAM酶属于SPASM亚家族的新发现。此外，我们发现辅助性[4Fe-4S]2+簇与三个Cys残基的硫原子和一个保守的Asp残基的羧基氧原子有罕见的连接。
PqqE is a radical S‐adenosyl‐l‐methionine (SAM) enzyme that catalyzes the initial reaction of pyrroloquinoline quinone (PQQ) biosynthesis. PqqE belongs to the SPASM (subtilosin/PQQ/anaerobic sulfatase/mycofactocin maturating enzymes) subfamily of the radical SAM superfamily and contains multiple Fe–S clusters. To characterize the Fe–S clusters in PqqE from Methylobacterium extorquens AM1, Cys residues conserved in the N‐terminal signature motif (CX 3 CX 2C) and the C‐terminal seven‐cysteine motif (CX 9–15 GX 4 CX n CX 2 CX 5 CX 3 CX nC; n = an unspecified number) were individually or simultaneously mutated into Ser. Biochemical and Mössbauer spectral analyses of as‐purified and reconstituted mutant enzymes confirmed the presence of three Fe–S clusters in PqqE: one [4Fe–4S]2+ cluster at the N‐terminal region that is essential for the reductive homolytic cleavage of SAM into methionine and 5′‐deoxyadenosyl radical, and one each [4Fe–4S]2+ and [2Fe–2S]2+ auxiliary clusters in the C‐terminal SPASM domain, which are assumed to serve for electron transfer between the buried active site and the protein surface. The presence of [2Fe–2S]2+ cluster is a novel finding for radical SAM enzyme belonging to the SPASM subfamily. Moreover, we found uncommon ligation of the auxiliary [4Fe–4S]2+ cluster with sulfur atoms of three Cys residues and a carboxyl oxygen atom of a conserved Asp residue.
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