Conversion of bacterial allosteric L-lactate dehydrogenases to constitutively active enzymes

对于LCLDH，S67 E、N68 D、E178 K和A235 K替代物相加地增加了不依赖于FBP的活性，降低了底物Km值。 X射线晶体学分析表明，这些突变在Q轴相关亚基之间引入了亚基间盐桥。 对于LPLDH，D 68 N和D 68 H替换一致地将双曲线基质饱和度曲线改变为S形。在TCLDH的情况下，L 67 E、N68 D、E178 K和A235 K（或A235 R）替换（Q突变）在不存在FBP的情况下仅略微改善底物Km，尽管它们改善了Vm值，而R173 Q和R216 L替换（P突变）显著改善了底物Km。 P-轴和Q-轴突变相加地改善了不依赖于FBP的活性和热稳定性。 结构比较表明，LCLDH和TCLDH表现出非常不同的结构变化，因此需要不同的设计，引入组成型高催化活性。

For LCLDH, S67E, N68D, E178K and A235K replacements additively increased the FBP-independent activity, reducing the substrate Km values. The X-ray crystallography indicated that these mutations introduced the inter-subunit salt bridges between the Q-axis related subunits. For LPLDH, D68N and D68H replacements consistently changed the hyperbolic substrate saturation curves to the sigmoidal ones. In the case of TCLDH, L67E, N68D, E178K and A235K (or A235R) replacements (Q mutations) only slightly improved substrate Km in the absence of FBP, though they improved Vm values, instead R173Q and R216L replacements (P mutations) markedly improved the substrate Km. The P-axis and Q-axis mutations additively improved the FBP-independent activity and thermal stability. Structural comparison indicated that LCLDH and TCLDH exhibit greatly different structural changes, and therefore require the different designs that introduce the constitutively high catalytic activities.