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Molecular Dynamics Analysis of a Rationally Designed Aldehyde Dehydrogenase Gives Insights into Improved Activity for the Non-Native Cofactor NAD.
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2020-04-07 , DOI: 10.1021/acssynbio.9b00527
Tobias J Gmelch 1 , Josef M Sperl 1 , Volker Sieber 1, 2, 3, 4
Affiliation  

The aldehyde dehydrogenase from Thermoplasma acidophilum was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols. In order to engineer the enzyme's cofactor specificity from NADP+ to NAD+, we identified selectivity-determining residues with the CSR-SALAD tool and investigated further positions based on the crystal structure. Stepwise combination of the initially discovered six point mutations allowed us to monitor the cross effects of each mutation, resulting in a final variant with reduced KM for the non-native cofactor NAD+ (from 18 to 0.6 mM) and an increased activity for the desired substrate d-glyceraldehyde (from 0.4 to 1.5 U/mg). Saturation mutagenesis of the residues at the entrance of the substrate pocket could eliminate substrate inhibition. Molecular dynamics simulations showed a significant gain of flexibility at the cofactor binding site for the final variant. The concomitant increase in stability against isobutanol and only a minor reduction in its temperature stability render the final variant a promising candidate for future optimization of our synthetic cell-free enzymatic cascade.

中文翻译:

合理设计的醛脱氢酶的分子动力学分析提供了对非天然辅因子NAD活性提高的见解。

嗜酸嗜热菌的醛脱氢酶以前被用作生产醇的无细胞合成反应级联反应中的关键酶。为了工程化酶从NADP +到NAD +的辅因子特异性,我们使用CSR-SALAD工具鉴定了决定选择性的残基,并基于晶体结构研究了进一步的位置。最初发现的6个点突变的逐步组合使我们能够监测每个突变的交叉效应,从而产生最终变体,其非天然辅因子NAD +的KM降低(从18到0.6 mM),而所需底物的活性增加d-甘油醛(0.4至1.5 U / mg)。底物袋入口处残留物的饱和诱变可消除底物抑制。分子动力学模拟表明,最终变体在辅因子结合位点具有显着的灵活性。随之而来的是对异丁醇的稳定性增加,而其温度稳定性仅有很小的降低,这使得最终的变体成为未来优化合成的无细胞酶促级联反应的有希望的候选者。
更新日期:2020-04-23
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