Institute of Biological Chemistry, Washington State University, Pullman, Washington, USA.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, USA.
mBio. 2024 Feb 14;15(2):e0298723. doi: 10.1128/mbio.02987-23. Epub 2023 Dec 21.
Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria and share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.
丙酮羧化酶（ACs）催化金属和 ATP 依赖性的丙酮和碳酸氢盐转化为乙酰乙酸盐。有趣的是，已经报道了两种具有生物化学特征的同源 ACs 具有不同的金属补体，这暗示了催化过程中不同的金属依赖性。来自变形菌门的 ACs 共享 68%的序列同一性，但据推测具有不同的催化金属。在这项工作中，两种 ACs 在相同条件下在 中表达，并进行了与 Mn(II)或 Fe(II)的螯合和重组实验的平行实验。电子顺磁共振和穆斯堡尔光谱学分别鉴定了活性位点结合的 Mn(II)或 Fe(II)的特征。这些实验表明，各自的 ACs 在没有伴侣蛋白、第二金属位点或翻译后修饰的帮助下，能够正确地进行金属掺入，尽管 Fe(II)具有预期的热力学优势，但每种 ACs 都偏爱一种独特的金属。尽管任何一种金属都有可能发挥拟议的路易斯酸性作用，但催化作用同样与同源金属独特相关。蛋白质结构的细微差异被认为是 Mn(II)或 Fe(II)的选择性过滤器。重要性欧文-威廉姆斯系列是指过渡金属配合物的预测稳定性，其中观察到的二价第一过渡金属配合物的总体稳定性在整个周期中增加。丙酮羧化酶（ACs）在催化碳酸氢盐和丙酮转化为乙酰乙酸盐的过程中，在其活性部位使用配位的二价金属。高度同源的 ACs 在其活性部位区分不同的二价金属，使得酶的变体更喜欢 Mn(II)而不是 Fe(II)，这与欧文-威廉姆斯预测的行为相违背。定义在第一过渡金属中促进金属区分的决定因素对于理解金属介导的催化和金属催化剂设计具有广泛的基础重要性。
