Department of Chemical Engineering, University of Washington, Seattle, Washington, USA.
CAS Key Laboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China.
mBio. 2018 Mar 27;9(2):e02430-17. doi: 10.1128/mBio.02430-17.
A recent surprising discovery of the activity of rare earth metals (lanthanides) as enzyme cofactors as well as transcriptional regulators has overturned the traditional assumption of biological inertia of these metals. However, so far, examples of such activities have been limited to alcohol dehydrogenases. Here we describe the physiological effects of a mutation in , a gene encoding a novel cytochrome, XoxG(4), and compare these to the effects of mutation in XoxF, a lanthanide-dependent methanol dehydrogenase, at the enzyme activity level and also at the community function level, using sp. strain LW13 as a model organism. Through comparative phenotypic characterization, we establish XoxG as the second protein directly involved in lanthanide-dependent metabolism, likely as a dedicated electron acceptor from XoxF. However, mutation in XoxG caused a phenotype that was dramatically different from the phenotype of the mutant in XoxF, suggesting a secondary function for this cytochrome, in metabolism of methane. We also purify XoxG(4) and demonstrate that this protein is a true cytochrome , based on the typical absorption spectra, and we demonstrate that XoxG can be directly reduced by a purified XoxF, supporting one of its proposed physiological functions. Overall, our data continue to suggest the complex nature of the interplay between the calcium-dependent and lanthanide-dependent alcohol oxidation systems, while they also suggest that addressing the roles of these alternative systems is essential at the enzyme and community function level, in addition to the gene transcription level. The lanthanide-dependent biochemistry of living organisms remains a barely tapped area of knowledge. So far, only a handful of lanthanide-dependent alcohol dehydrogenases have been described, and their regulation by lanthanides has been demonstrated at the transcription level. Little information is available regarding the concentrations of lanthanides that could support sufficient enzymatic activities to support specific metabolisms, and so far, no other redox proteins involved in lanthanide-dependent methanotrophy have been demonstrated. The research presented here provides enzyme activity-level data on lanthanide-dependent methanotrophy in a model methanotroph. Additionally, we identify a second protein important for lanthanide-dependent metabolism in this organism, XoxG(4), a novel cytochrome. XoxG(4) appears to have multiple functions in methanotrophy, one function as an electron acceptor from XoxF and another function remaining unknown. On the basis of the dramatic phenotype of the XoxG(4) mutant, this function must be crucial for methanotrophy.
最近，人们发现稀土金属（镧系元素）不仅可以作为酶辅助因子，还可以作为转录调节剂，这一惊人的发现颠覆了人们对这些金属在生物体内惰性的传统认识。然而，到目前为止，这种活性的例子仅限于醇脱氢酶。在这里，我们描述了一个基因的突变对 的影响，该基因编码一种新型细胞色素 XoxG(4)，并将其与突变的影响进行比较 XoxF，一种依赖镧系元素的甲醇脱氢酶，在酶活性水平和群落功能水平上，使用 sp. 菌株 LW13 作为模式生物。通过比较表型特征，我们确定 XoxG 是第二个直接参与镧系元素代谢的蛋白质，可能是 XoxF 的专用电子受体。然而，XoxG 的突变导致的表型与 XoxF 突变体的表型截然不同，这表明该细胞色素可能具有甲烷代谢的次要功能。我们还纯化了 XoxG(4)，并证明根据典型的吸收光谱，该蛋白是一种真正的细胞色素 ，我们还证明 XoxG 可以被纯化的 XoxF 直接还原，支持其生理功能之一。总的来说，我们的数据继续表明钙依赖性和镧依赖性醇氧化系统之间相互作用的复杂性，同时也表明在基因转录水平之外，还需要在酶和群落功能水平上解决这些替代系统的作用。生物体的镧依赖性生物化学仍然是一个几乎未被开发的知识领域。到目前为止，只有少数几种依赖镧系元素的醇脱氢酶被描述过，它们的镧系元素调节作用已经在转录水平上得到了证明。关于支持特定代谢所需的镧系元素的浓度的信息很少，到目前为止，还没有其他参与镧系元素依赖型甲烷氧化作用的氧化还原蛋白被证明。本研究提供了模型甲烷营养菌中镧系元素依赖型甲烷氧化作用的酶活性水平数据。此外，我们在该生物体内确定了另一种对镧系元素依赖型代谢很重要的蛋白质 XoxG(4)，一种新型细胞色素。XoxG(4)似乎在甲烷氧化作用中有多种功能，一种功能是作为 XoxF 的电子受体，另一种功能尚不清楚。基于 XoxG(4)突变体的显著表型，这个功能对甲烷氧化作用至关重要。
