Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
Department of Biochemistry, Michigan State University, East Lansing, Michigan, USA.
J Biol Chem. 2020 Jun 12;295(24):8272-8284. doi: 10.1074/jbc.RA120.013227. Epub 2020 May 4.
The lanthanide elements (Ln), those with atomic numbers 57-63 (excluding promethium, Pm), 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 Ca-PQQ cofactor. An important missing piece of the Ln puzzle is defining what features distinguish enzymes that use Ln-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium AM1, 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 bound in the active-site region and coordinated by Asp Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp is needed for catalytic function, activity, and La coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La, coordinated Ca 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 La These results provide evidence that a Ln-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 metabolism.
镧系元素（Ln）是原子序数为 57-63 的元素（不包括钷，Pm），它们在细菌 XoxF 甲醇脱氢酶（MDHs）和 ExaF 乙醇脱氢酶（EDHs）中与吡咯并喹啉醌（PQQ）形成辅助因子复合物，扩大了生物元素的范围，并开辟了新的代谢和生态学领域。其他 MDHs，称为 MxaFIs，在序列和结构上与这些蛋白质相关，但它们具有 Ca-PQQ 辅助因子。Ln 之谜的一个重要缺失部分是定义区分使用 Ln-PQQ 辅助因子的酶和不使用 Ln-PQQ 辅助因子的酶的特征。在这里，我们使用模型甲基营养细菌 AM1 的 XoxF1 MDH 研究了一个提议的镧系元素配位天冬氨酸残基的功能重要性。我们报告了 XoxF1 的两个晶体结构，一个带有 PQQ，另一个没有 PQQ，都在活性部位区域结合 La，由 Asp 配位。使用构建体生产重组 XoxF1 或其 D320A 变体，我们表明 Asp 对于催化功能、活性和 La 配位是必需的。当在没有 La 的情况下产生 XoxF1 和 XoxF1 D320A 时，它们配位 Ca，但表现出很少或没有催化活性。我们还在 ExaF 中生成了平行取代，生成了 ExaF D319S，并发现该变体失去了在 La 存在下有效氧化乙醇的能力。这些结果提供了证据，证明 Ln 配位的天冬氨酸对于 XoxF MDHs 和 ExaF EDHs 的酶促功能是必不可少的，支持了这些酶的序列以及编码它们的基因是 Ln 代谢标志物的观点。
