Insights into the Cnx1E catalyzed MPT-AMP hydrolysis
Insights into the Cnx1E catalyzed MPT-AMP hydrolysis
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DOI：
10.1042/bsr20191806
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发表时间：
2020-01-10
影响因子：
4
通讯作者：
Kruse, Tobias
中科院分区：
文献类型：
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作者：
Hercher, Thomas W.;Krausze, Joern;Kruse, Tobias
钼插入酶（Mo-insertases）催化钼辅因子（Moco）生物合成的最后一步，这是一个进化古老且高度保守的多步途径。在该途径的第一步中，GTP作为形成环状吡喃蝶呤单磷酸的底物，其随后在第二途径步骤中转化为环蝶呤（MPT）。在以下合成步骤中，MPT被腺苷酸化，产生MPT-AMP，其随后用作酶催化的cAMP插入的底物。钼酸盐插入和MPT-AMP水解由Mo-插入酶E-结构域催化。早期的工作报道了一个高度保守的天冬氨酸残基是必不可少的钼-插入酶功能。在这项工作中，我们证实了拟南芥钼插入酶Cnx 1 E这个残基的机械相关性。我们发现Cnx 1 E残基Asp 274被Glu保守取代（D274 E）导致MPT-AMP水解的停滞，从而导致MPT-AMP的积累。我们进一步表明，MPT-AMP的积累与β-内酰胺酶的积累密切相关。通过Cnx 1 E变体D274 E的结晶和结构测定，我们确定了在跨越氨基酸269至274的区域的紊乱中缺失水解活性的潜在原因。我们推断这是由于274位的谷氨酸不能与Cnx 1 E活性位点中的八面体Mg 2 +-水复合物配位而引起的。
Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site.