Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions
Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions
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DOI：
10.1042/bj20140895
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发表时间：
2014-11-15
影响因子：
4.1
通讯作者：
Roach, Peter L.
中科院分区：
文献类型：
--
作者：
Harmer, Jenny E.;Hiscox, Martyn J.;Roach, Peter L.
脂酰辅因子对生物体来说是必不可少的，由两个硫原子插入辛酰基底物相对不活跃的C-H键而产生。该反应需要S-腺苷甲硫氨酸自由基超家族成员的硫酰合成酶。在本研究中，我们解决了硫辛基合成酶的晶体结构，其中两个[4Fe-4S]簇结合在TIM桶的两端，这是自由基SAM超家族的常见折叠。还原SAM裂解所需的簇保留了自由基SAM超家族的特征，但辅助簇被硫酰合成酶特有的CX4CX5C基序结合。辅助簇的第四个配体是一种极不寻常的丝氨酸残基。定点突变表明，这种保守的丝氨酸配体对于硫插入步骤是必不可少的。一个结晶化的硫酰合成酶(LIPA)复合体含有5‘-甲硫基腺苷(MTA)，它是SAM的分解产物，结合在可能的SAM结合部位。模型已经确定了一个18埃(1埃=0.1纳米)深的通道，该通道的比例很好，可以容纳辛酰基底物。这些结果表明，辅助簇可能是硫的供体，但进入第二次硫插入反应的硫离子需要从辅助簇中失去一个铁原子，这可能是丝氨酸配体所能实现的。
Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized lipoyl synthase (LipA) complex contains 5'-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modelling has identified an 18 angstrom (1 angstrom=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable.