Converting NAD-specific inositol dehydrogenase to an efficient NADP-selective catalyst, with a surprising twist.
Converting NAD-specific inositol dehydrogenase to an efficient NADP-selective catalyst, with a surprising twist.
复制标题
将 NAD 特异性肌醇脱氢酶转化为有效的 NADP 选择性催化剂，具有令人惊讶的转折。
作者：
Hongyan Zheng;D. Bertwistle;D. Sanders;D. R. Palmer
来自枯草芽孢杆菌的肌醇脱氢酶（IDH，EC 1.1.1.18）将肌醇转化为鲨肌醇，并且其活性严格依赖于NAD。我们试图改变辅酶特异性以产生NADP依赖性酶，以增强我们对辅酶选择性的理解，并创造一种能够在生物催化过程中回收NADP的酶。检查可用的结构信息相关的GFO/莫卡/IDH家族的辅酶选择性和先例改变允许我们选择残基的取代，并构建了9个单，双，和三重突变体。用B进行诱变实验。枯草杆菌IDH证明是非常成功的;双突变体D35 S/V36 R优选NADP到NAD的5倍。该突变体是一个优良的催化剂，其NADP的二级速率常数为370 000 s，三突变体A12 K/D35 S/V36 R的二级速率常数为570 000 s，高于野生型IDH与NAD的二级速率常数。双突变体A12 K/D35 S的高分辨率X射线晶体结构在与NADP的复合物中得到解决。令人惊讶的是，辅酶的结合被改变，使得尽管烟酰胺环保持催化所需的位置，但辅酶已经扭曲了近90°，因此腺嘌呤部分不再与野生型酶中的罗斯曼折叠中的疏水裂缝结合。这种结合构象的变化以前在突变的腺苷酸酶中没有观察到。
myo-Inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis converts myo-inositol to scyllo-inosose and is strictly dependent on NAD for activity. We sought to alter the coenzyme specificity to generate an NADP-dependent enzyme in order to enhance our understanding of coenzyme selectivity and to create an enzyme capable of recycling NADP in biocatalytic processes. Examination of available structural information related to the GFO/MocA/IDH family of dehydrogenases and precedents for altering coenzyme selectivity allowed us to select residues for substitution, and nine single, double, and triple mutants were constructed. Mutagenesis experiments with B. subtilis IDH proved extremely successful; the double mutant D35S/V36R preferred NADP to NAD by a factor of 5. This mutant is an excellent catalyst with a second-order rate constant with respect to NADP of 370 000 s⁻¹ M⁻¹, and the triple mutant A12K/D35S/V36R had a value of 570 000 s⁻¹ M⁻¹, higher than that of the wild-type IDH with NAD. The high-resolution X-ray crystal structure of the double mutant A12K/D35S was solved in complex with NADP. Surprisingly, the binding of the coenzyme is altered such that although the nicotinamide ring maintains the required position for catalysis, the coenzyme has twisted by nearly 90°, so the adenine moiety no longer binds to a hydrophobic cleft in the Rossmann fold as in the wild-type enzyme. This change in binding conformation has not previously been observed in mutated dehydrogenases.
枯草芽孢杆菌肌醇脱氢酶 (IDH) 的纯化、结晶和初步 X 射线分析。
DOI：
10.1107/s1744309108000328
发表时间：
2008
期刊：
Acta crystallographica. Section F, Structural biology and crystallization communications
影响因子：
--
作者：
VanStraaten,KE;Hoffort,A;Palmer,DRJ;Sanders,DAR
通讯作者：
Sanders,DAR
一致。
DOI：
10.1097/iae.0000000000001602
发表时间：
2017
期刊：
Retina (Philadelphia, Pa.)
影响因子：
--
作者：
Todorich,Bozho;Thanos,Aristomenis;Yonekawa,Yoshihiro;Thomas,BenjaminJ;Faia,LisaJ;Chang,Emmanuel;Shulman,Julia;Olsen,KarlR;Blair,MichaelP;Shapiro,MichaelP;Ferrone,Philip;Vajzovic,Lejla;Toth,CynthiaA;Lee,ThomasC;Robinson,
通讯作者：
Robinson,
DOI：
10.1093/protein/15.2.131
发表时间：
2002-02-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Banta, S;Swanson, BA;Anderson, S
通讯作者：
Anderson, S