Structural Study on the Reaction Mechanism of a Free Serine Kinase Involved in Cysteine Biosynthesis
Structural Study on the Reaction Mechanism of a Free Serine Kinase Involved in Cysteine Biosynthesis
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半胱氨酸生物合成中游离丝氨酸激酶反应机制的结构研究
DOI：
10.1021/acschembio.7b00064
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发表时间：
2017
影响因子：
4
通讯作者：
Kunio Miki
中科院分区：
文献类型：
--
作者：
Ryuhei Nagata;Masahiro Fujihashi;Hiroki Kawamura;Takaaki Sato;Takayuki Fujita;Haruyuki Atomi;Kunio Miki
超嗜热古菌Thermococcus kodakarensis中的一种游离丝氨酸激酶（SerK）参与L-半胱氨酸的生物合成。该酶将ADP和L-丝氨酸（Ser）转化为AMP和O-磷酸-L-丝氨酸（Sep），后者是半胱氨酸的前体。SerK是第一个被鉴定的磷酸化游离丝氨酸的酶，而丝氨酸/苏氨酸蛋白激酶已经被很好地研究。SerK与已知激酶的序列相似性较低，表明其反应机制与已知激酶不同。在这里，我们确定了SerK从T的晶体结构。kodakarensis（Tk-SerK）。总体结构分为两个域。在AMP复合物和ADP复合物中的两个结构域之间发现大的裂缝。该裂缝在三元产物复合物（Sep、AMP和Tk-SerK）中闭合，并且也可能在三元底物复合物（Ser、ADP和Tk-SerK）中闭合。E30的封闭作用可能使E30的羧基重新定位在Ser的Oγ原子附近，Oγ原子被E30去质子化并攻击ADP的β-磷酸形成SEP，E30 A突变体酶活性的显著降低与此机制相一致。我们的结构还揭示了有助于配体结合的残基。这些残基在细菌的未表征蛋白质中的保守性可能会提高不仅在古细菌中而且在细菌中存在游离Ser激酶的可能性。
A free serine kinase (SerK) is involved inl-cysteine biosynthesis in the hyperthermophilic archaeonThermococcus kodakarensis. The enzyme converts ADP andl-serine (Ser) into AMP andO-phospho-l-serine (Sep), which is a precursor ofl-cysteine. SerK is the first identified enzyme that phosphorylates free serine, while serine/threonine protein kinases have been well studied. SerK displays low sequence similarities to known kinases, suggesting that its reaction mechanism is different from those of known kinases. Here, we determined the crystal structures of SerK fromT. kodakarensis(Tk-SerK). The overall structure is divided into two domains. A large cleft is found between the two domains in the AMP complex and in the ADP complex. The cleft is closed in the ternary product complex (Sep, AMP, andTk-SerK) and may also be in the ternary substrate complex (Ser, ADP, andTk-SerK). The closure may reorient the carboxyl group of E30 near to the Oγ atom of Ser. The Oγ atom is considered to be deprotonated by E30 and to attack the β-phosphate of ADP to form Sep. The substantial decrease in the activity of the E30A mutant is consistent with this mechanism. Our structures also revealed the residues that contribute to the ligand binding. The conservation of these residues in uncharacterized proteins from bacteria may raise the possibility of the presence of free Ser kinases not only in archaea but also in bacteria.