Data_Sheet_1_Interrogating the Role of the Two Distinct Fructose-Bisphosphate Aldolases of Bacillus methanolicus by Site-Directed Mutagenesis of Key Amino Acids and Gene Repression by CRISPR Interference.pdf

The Gram-positive Bacillus methanolicus shows plasmid-dependent methylotrophy. This facultative ribulose monophosphate (RuMP) cycle methylotroph possesses two fructose bisphosphate aldolases (FBA) with distinct kinetic properties. The chromosomally encoded FBAC is the major glycolytic aldolase. The gene for the major gluconeogenic aldolase FBAP is found on the natural plasmid pBM19 and is induced during methylotrophic growth. The crystal structures of both enzymes were solved at 2.2 Å and 2.0 Å, respectively, and they suggested amino acid residue 51 to be crucial for binding fructose-1,6-bisphosphate (FBP) as substrate and amino acid residue 140 for active site zinc atom coordination. As FBAC and FBAP differed at these positions, site-directed mutagenesis (SDM) was performed to exchange one or both amino acid residues of the respective proteins. The aldol cleavage reaction was negatively affected by the amino acid exchanges that led to a complete loss of glycolytic activity of FBAP. However, both FBAC and FBAP maintained gluconeogenic aldol condensation activity, and the amino acid exchanges improved the catalytic efficiency of the major glycolytic aldolase FBAC in gluconeogenic direction at least 3-fold. These results confirmed the importance of the structural differences between FBAC and FBAP concerning their distinct enzymatic properties. In order to investigate the physiological roles of both aldolases, the expression of their genes was repressed individually by CRISPR interference (CRISPRi). The fbaC RNA levels were reduced by CRISPRi, but concomitantly the fbaP RNA levels were increased. Vice versa, a similar compensatory increase of the fbaC RNA levels was observed when fbaP was repressed by CRISPRi. In addition, targeting fbaP decreased tktP RNA levels since both genes are cotranscribed in a bicistronic operon. However, reduced tktP RNA levels were not compensated for by increased RNA levels of the chromosomal transketolase gene tktC.

革兰氏阳性甲醇芽孢杆菌（Bacillus methanolicus）具有依赖质粒的甲基营养生长特性。该兼性核酮糖单磷酸（RuMP）循环型甲基营养菌拥有两种果糖双磷酸醛缩酶（FBA），二者具备截然不同的动力学特性。其中，染色体编码的FBAC为主要糖酵解型醛缩酶；而主要糖异生型醛缩酶FBAP的编码基因位于天然质粒pBM19上，并在甲基营养型生长过程中被诱导表达。两种酶的晶体结构分别以2.2 Å和2.0 Å的分辨率解析完成，结构分析显示，第51位氨基酸残基对结合作为底物的果糖-1,6-双磷酸（FBP）至关重要，第140位氨基酸残基则负责活性位点的锌原子配位。由于FBAC与FBAP在上述两个位点存在氨基酸差异，本研究通过定点诱变（SDM）技术对两种酶的单个或两个氨基酸残基进行了替换。实验结果显示，氨基酸替换对醛缩裂解反应产生了负面影响，致使FBAP完全丧失了糖酵解活性；但FBAC与FBAP均保留了糖异生型的醛缩缩合活性，且氨基酸替换使主要糖酵解型醛缩酶FBAC在糖异生方向的催化效率至少提升了3倍。上述结果证实了FBAC与FBAP之间的结构差异是二者酶学特性截然不同的关键原因。为探究两种醛缩酶的生理功能，本研究通过CRISPR干扰（CRISPRi）技术分别抑制了它们的编码基因表达：当通过CRISPRi抑制fbaC的表达时，fbaC的RNA水平降低，但与此同时fbaP的RNA水平出现代偿性升高；反之，当抑制fbaP的表达时，也观察到了类似的fbaC RNA水平代偿性升高现象。此外，由于fbaP与tktP以双顺反子操纵子的形式共转录，靶向fbaP的CRISPRi会导致tktP的RNA水平降低，但染色体编码的转酮醇酶基因tktC的RNA水平并未出现代偿性升高以弥补tktP RNA水平的下调。