Protein residues that control the reaction trajectory in S-adenosylmethionine radical enzymes: mutagenesis of asparagine 153 and aspartate 155 in Escherichia coli biotin synthase.
Protein residues that control the reaction trajectory in S-adenosylmethionine radical enzymes: mutagenesis of asparagine 153 and aspartate 155 in Escherichia coli biotin synthase.
复制标题
DOI：
10.1021/bi8022569
复制
发表时间：
2009-03-24
期刊：
影响因子：
2.9
通讯作者：
Jarrett, Joseph T.
中科院分区：
文献类型：
--
作者：
Farrar, Christine E.;Jarrett, Joseph T.
生物素合酶催化硫原子氧化加成到脱硫生物素 (DTB) 上，生成生物素噻吩环。该反应由 S-腺苷-L-甲硫氨酸 (AdoMet) 的锍中心还原裂解引发，生成甲硫氨酸和瞬态 5'-脱氧腺苷自由基，该自由基通过从 DTB 中夺取氢原子而起到氧化剂的作用。生物素合酶含有高度保守的序列基序YNHNLD，其中Asn153和Asp155与AdoMet的核糖羟基形成氢键。在目前的工作中，我们构建了四个单独的定点突变来改变这两个残基中的每一个，以探测它们在活性位点中的作用。我们使用分子量过滤测定表明，对于大多数突变酶，底物的结合仅受到轻微影响。体外测定表明，几种突变酶能够还原性裂解 AdoMet，但没有一种能够产生大量的生物素。一些突变体，尤其是 Asn153Ser，能够产生高水平的稳定中间体 9-巯基脱硫生物素。一些突变体，例如 Asp155Asn 和 Asn153Ala，产生了一种替代产物，通过质谱分析初步鉴定为 5'-巯基-5'-脱氧腺苷，它是通过直接攻击 [4Fe-4S]2+ 簇上的 5'-脱氧腺苷基自由基而产生的。总的来说，这些结果表明，与 AdoMet 和 DTB 形成氢键的蛋白质残基对于在催化循环期间保留中间体以及针对 5'-脱氧腺苷基自由基的反应性非常重要。
Biotin synthase catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This reaction is initiated by the reductive cleavage of the sulfonium center of S-adenosyl-L-methionine (AdoMet), generating methionine and a transient 5′-deoxyadenosyl radical that functions as an oxidant by abstracting hydrogen atoms from DTB. Biotin synthase contains a highly conserved sequence motif, YNHNLD, in which Asn153 and Asp155 form hydrogen bonds with the ribose hydroxyl groups of AdoMet. In the present work, we constructed four individual site-directed mutations to change each of these two residues in order to probe their role in the active site. We used molecular weight filtration assays to show that for most of the mutant enzymes, binding of the substrates was only slightly affected. In vitro assays demonstrate that several of the mutant enzymes were able to reductively cleave AdoMet, but none were able to produce a significant amount of biotin. Several of the mutants, especially Asn153Ser, were able to produce high levels of the stable intermediate 9-mercaptodethiobiotin. Some of the mutants, such as Asp155Asn and Asn153Ala, produced instead an alternate product tentatively identified by mass spectrometry as 5′-mercapto-5′-deoxyadenosine, generated by direct attack of the 5′-deoxyadenosyl radical on the [4Fe-4S]2+ cluster. Collectively these results suggest that the protein residues that form hydrogen bonds to AdoMet and DTB are important for retaining intermediates during the catalytic cycle and for targeting the reactivity of the 5′-deoxydenosyl radical.
登录
查看更多内容
DOI：
10.1006/bbrc.2000.2313
发表时间：
2000-03-16
影响因子：
3.1
作者：
Broderick, JB;Henshaw, TF;Broderick, WE
通讯作者：
Broderick, WE
影响因子：
14.9
作者：
Sofia, HJ;Chen, G;Miller, NE
通讯作者：
Miller, NE
影响因子：
2.9
作者：
Duin, EC;Lafferty, ME;Johnson, MK
通讯作者：
Johnson, MK
影响因子：
14.9
作者：
Nicolet, Y;Drennan, CL
通讯作者：
Drennan, CL
影响因子：
56.9
作者：
Berkovitch, F;Nicolet, Y;Drennan, CL
通讯作者：
Drennan, CL