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Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle.
BMC Microbiology ( IF 4.0 ) Pub Date : 2020-03-24 , DOI: 10.1186/s12866-020-01750-6
Johannes Pfeifenschneider 1 , Benno Markert 1 , Jessica Stolzenberger 1 , Trygve Brautaset 2 , Volker F Wendisch 1
Affiliation  

The Gram-positive facultative methylotrophic bacterium Bacillus methanolicus uses the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle for growth on the C1 carbon source methanol. Previous genome sequencing of the physiologically different B. methanolicus wild-type strains MGA3 and PB1 has unraveled all putative RuMP cycle genes and later, several of the RuMP cycle enzymes of MGA3 have been biochemically characterized. In this study, the focus was on the characterization of the transaldolase (Ta) and its possible role in the RuMP cycle in B. methanolicus. The Ta genes of B. methanolicus MGA3 and PB1 were recombinantly expressed in Escherichia coli, and the gene products were purified and characterized. The PB1 Ta protein was found to be active as a homodimer with a molecular weight of 54 kDa and displayed KM of 0.74 mM and Vmax of 16.3 U/mg using Fructose-6 phosphate as the substrate. In contrast, the MGA3 Ta gene, which encodes a truncated Ta protein lacking 80 amino acids at the N-terminus, showed no Ta activity. Seven different mutant genes expressing various full-length MGA3 Ta proteins were constructed and all gene products displayed Ta activities. Moreover, MGA3 cells displayed Ta activities similar as PB1 cells in crude extracts. While it is well established that B. methanolicus can use the SBPase variant of the RuMP cycle this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP.

中文翻译:

甲醇芽孢杆菌中的转醛缩酶:核糖一磷酸循环的生化特征和生物学作用。

革兰氏阳性兼性甲基营养型甲醇芽孢杆菌使用核糖单磷酸(RuMP)循环的sedoheptulose-1,7-bisphosphatase(SBPase)变体在C1碳源甲醇上生长。先前生理学上不同的甲醇毕赤酵母野生型菌株MGA3和PB1的基因组测序已揭示了所有推定的RuMP循环基因,后来,已对MGA3的几种RuMP循环酶进行了生化鉴定。在这项研究中,重点是转醛醇酶(Ta)的表征及其在甲醇双歧杆菌RuMP循环中的可能作用。甲醇芽孢杆菌MGA3和PB1的Ta基因在大肠杆菌中重组表达,并纯化和鉴定了基因产物。发现PB1 Ta蛋白具有同二聚体的活性,分子量为54 kDa,使用果糖-6磷酸酯作为底物显示KM为0.74 mM,Vmax为16.3 U / mg。相比之下,MGA3 Ta基因编码的Ta活性没有,该基因在N端缺少80个氨基酸的截短的Ta蛋白。构建了七个表达各种全长MGA3 Ta蛋白的不同突变基因,所有基因产物均显示出Ta活性。此外,MGA3细胞在粗提物中显示出与PB1细胞相似的Ta活性。众所周知,甲醇芽孢杆菌可以使用RuMP循环的SBPase变异体,但这项研究表明甲醇芽孢杆菌具有Ta活性,也可以运行RuMP的Ta变异体。MGA3 Ta基因编码一个在N末端缺少80个氨基酸的截短的Ta蛋白,但没有Ta活性。构建了七个表达各种全长MGA3 Ta蛋白的不同突变基因,所有基因产物均显示出Ta活性。此外,MGA3细胞在粗提物中显示出与PB1细胞相似的Ta活性。尽管已经确定甲醇芽孢杆菌可以使用RuMP循环的SBPase变体,但这项研究表明,甲醇芽孢杆菌具有Ta活性,也可以运行RuMP的Ta变体。MGA3 Ta基因编码一个在N末端缺少80个氨基酸的截短的Ta蛋白,但没有Ta活性。构建了七个表达各种全长MGA3 Ta蛋白的不同突变基因,所有基因产物均显示出Ta活性。此外,MGA3细胞在粗提物中显示出与PB1细胞相似的Ta活性。众所周知,甲醇芽孢杆菌可以使用RuMP循环的SBPase变异体,但这项研究表明甲醇芽孢杆菌具有Ta活性,也可以运行RuMP的Ta变异体。MGA3细胞在粗提物中显示出与PB1细胞相似的Ta活性。众所周知,甲醇芽孢杆菌可以使用RuMP循环的SBPase变异体,但这项研究表明甲醇芽孢杆菌具有Ta活性,也可以运行RuMP的Ta变异体。MGA3细胞在粗提物中显示出与PB1细胞相似的Ta活性。尽管已经确定甲醇芽孢杆菌可以使用RuMP循环的SBPase变体,但这项研究表明,甲醇芽孢杆菌具有Ta活性,也可以运行RuMP的Ta变体。
更新日期:2020-04-22
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