Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function
Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function
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DOI：
10.1074/jbc.ra120.013227
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发表时间：
2020-06-12
影响因子：
4.8
通讯作者：
Martinez-Gomez, N. Cecilia
中科院分区：
文献类型：
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作者：
Good, Nathan M.;Fellner, Matthias;Martinez-Gomez, N. Cecilia
稀土元素(Ln(3+))在细菌XoxF甲醇脱氢酶(MdHS)和ExaF乙醇脱氢酶(EDHS)中与吡咯喹啉(PQQ)形成辅因子络合物，拓宽了生物元素的范围，开辟了代谢和生态学的新领域。其他MdHs，称为MxaFIs，在序列和结构上与这些蛋白质相关，但它们却拥有一种钙-PQQ辅因子。Ln(3+)拼图中缺少的一个重要部分是定义使用Ln(3+)-PQQ辅因子的酶与不使用Ln(3+)-PQQ辅因子的酶的区别。在这里，使用来自模式甲基化营养细菌的XoxF1 MDH，我们研究了所提出的稀土配位天冬氨酸残基的功能重要性。我们报道了XoxF1的两种晶体结构，一种是有PQQ的，另一种是没有PQQ的，都是La(3+)结合在活性中心区域，并与Asp(320)配位。利用构建的重组XoxF1或其D320A变异体，我们证明了Asp(320)是活体催化功能、体外活性和La(3+)配位所必需的。XoxF1和XoxF1 D320A在无La3+的情况下产生配位Ca(2+)，但几乎没有催化活性。我们还在ExaF中产生了平行取代来产生ExaF D319S，并发现该变体失去了与La3+有效地氧化乙醇的能力。这些结果证明了Ln(3+)配位天冬氨酸对XoxF MdHS和ExaF EDHS的酶功能是必不可少的，支持了这些酶的序列和编码它们的基因是Ln(3+)代谢的标记的观点。
The lanthanide elements (Ln(3+)), those with atomic numbers 57?63 (excluding promethium, Pm3+), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln(3+)puzzle is defining what features distinguish enzymes that use Ln(3+)-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacteriumMethylorubrum extorquensAM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La(3+)bound in the active-site region and coordinated by Asp(320). Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp(320)is needed forin vivocatalytic function,in vitroactivity, and La(3+)coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinated Ca(2+)but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La3+. These results provide evidence that a Ln(3+)-coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln(3+)metabolism.