Culpepper Megen A, Rosenzweig Amy C
Departments of Molecular Biosciences and Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
Biochemistry. 2014 Oct 7;53(39):6211-9. doi: 10.1021/bi500850j. Epub 2014 Sep 19.
In the initial steps of their metabolic pathway, methanotrophic bacteria oxidize methane to methanol with methane monooxygenases (MMOs) and methanol to formaldehyde with methanol dehydrogenases (MDHs). Several lines of evidence suggest that the membrane-bound or particulate MMO (pMMO) and MDH interact to form a metabolic supercomplex. To further investigate the possible existence of such a supercomplex, native MDH from Methylococcus capsulatus (Bath) has been purified and characterized by size exclusion chromatography with multi-angle light scattering and X-ray crystallography. M. capsulatus (Bath) MDH is primarily a dimer in solution, although an oligomeric species with a molecular mass of ∼450-560 kDa forms at higher protein concentrations. The 2.57 Å resolution crystal structure reveals an overall fold and α2β2 dimeric architecture similar to those of other MDH structures. In addition, biolayer interferometry studies demonstrate specific protein-protein interactions between MDH and M. capsulatus (Bath) pMMO as well as between MDH and the truncated recombinant periplasmic domains of M. capsulatus (Bath) pMMO (spmoB). These interactions exhibit KD values of 833 ± 409 nM and 9.0 ± 7.7 μM, respectively. The biochemical data combined with analysis of the crystal lattice interactions observed in the MDH structure suggest a model in which MDH and pMMO associate not as a discrete, stoichiometric complex but as a larger assembly scaffolded by the intracytoplasmic membranes.
在其代谢途径的初始步骤中，甲烷营养细菌利用甲烷单加氧酶（MMOs）将甲烷氧化为甲醇，并利用甲醇脱氢酶（MDHs）将甲醇氧化为甲醛。多条证据表明，膜结合或颗粒状MMO（pMMO）和MDH相互作用形成代谢超复合物。为了进一步研究这种超复合物的可能存在，已对来自荚膜甲基球菌（巴斯）的天然MDH进行了纯化，并通过多角度光散射尺寸排阻色谱法和X射线晶体学对其进行了表征。荚膜甲基球菌（巴斯）MDH在溶液中主要是二聚体，尽管在较高蛋白质浓度下会形成分子量约为450 - 560 kDa的寡聚体。分辨率为2.57 Å的晶体结构揭示了其整体折叠和α2β2二聚体结构，与其他MDH结构相似。此外，生物层干涉测量研究表明，MDH与荚膜甲基球菌（巴斯）pMMO之间以及MDH与荚膜甲基球菌（巴斯）pMMO的截短重组周质结构域（spmoB）之间存在特异性蛋白质 - 蛋白质相互作用。这些相互作用的解离常数（KD）值分别为833 ± 409 nM和9.0 ± 7.7 μM。生化数据与MDH结构中观察到的晶格相互作用分析相结合，提出了一个模型，其中MDH和pMMO不是作为离散的化学计量复合物结合，而是作为由胞内膜搭建的更大组装体结合。
