Contrasting in vitro and in vivo methanol oxidation activities of lanthanide-dependent alcohol dehydrogenases XoxF1 and ExaF from Methylobacterium extorquens AM1
Contrasting in vitro and in vivo methanol oxidation activities of lanthanide-dependent alcohol dehydrogenases XoxF1 and ExaF from Methylobacterium extorquens AM1
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DOI：
10.1038/s41598-019-41043-1
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发表时间：
2019-03-12
影响因子：
4.6
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.;Moore, Riley S.;Martinez-Gomez, N. Cecilia
镧系元素作为辅助因子被xoxf型甲醇脱氢酶（MDHs）催化。一个主要的假设是XoxF酶通过甲醇氧化产生甲酸，这可能会影响需要甲醛同化的生物体。我们报告的遗传和表型证据表明，来自敲诈勒索甲基杆菌AM1的XoxF1 （MexAM1_1740）在甲醇中生长时产生甲醛，而不是甲酸。用镧或钕提纯的酶可以氧化甲醛。然而，通过2,6-二氯苯酚-吲哚酚（DCPIP）还原的甲醛氧化在野生型菌株甲醇和镧培养的无细胞提取物中未被检测到。甲醛激活酶（Fae）是Ln甲基营养生长所必需的，这表明xoxf1介导的甲醛产生是必不可少的。外源镧的加入使甲醇的生长速率提高了9-12%，但与甲醇消耗量或甲醛积累的变化无关。甲醇镧生长转录组学分析显示，xox1基因上调，mxa基因下调，与ln开关一致，甲醛转化基因无差异表达，吡罗喹啉醌（PQQ）生物合成基因下调，fdh4甲酸脱氢酶（FDH）基因上调。此外，当镧系元素存在时，ln依赖性乙醇脱氢酶ExaF降低了fae突变菌株的甲醇敏感性，这为ExaF在ln依赖性甲基化过程中起辅助作用的能力提供了证据。
Lanthanide (Ln) elements are utilized as cofactors for catalysis by XoxF-type methanol dehydrogenases (MDHs). A primary assumption is that XoxF enzymes produce formate from methanol oxidation, which could impact organisms that require formaldehyde for assimilation. We report genetic and phenotypic evidence showing that XoxF1 (MexAM1_1740) from Methylobacterium extorquens AM1 produces formaldehyde, and not formate, during growth with methanol. Enzyme purified with lanthanum or neodymium oxidizes formaldehyde. However, formaldehyde oxidation via 2,6-dichlorophenol-indophenol (DCPIP) reduction is not detected in cell-free extracts from wild-type strain methanol-and lanthanum-grown cultures. Formaldehyde activating enzyme (Fae) is required for Ln methylotrophic growth, demonstrating that XoxF1-mediated production of formaldehyde is essential. Addition of exogenous lanthanum increases growth rate with methanol by 9-12% but does not correlate with changes to methanol consumption or formaldehyde accumulation. Transcriptomics analysis of lanthanum methanol growth shows upregulation of xox1 and downregulation of mxa genes, consistent with the Ln-switch, no differential expression of formaldehyde conversion genes, downregulation of pyrroloquinoline quinone (PQQ) biosynthesis genes, and upregulation of fdh4 formate dehydrogenase (FDH) genes. Additionally, the Ln-dependent ethanol dehydrogenase ExaF reduces methanol sensitivity in the fae mutant strain when lanthanides are present, providing evidence for the capacity of an auxiliary role for ExaF during Ln-dependent methylotrophy.