Functional analysis of active amino acid residues of the mercaptosuccinate dioxygenase of Variovorax paradoxus B4
Functional analysis of active amino acid residues of the mercaptosuccinate dioxygenase of Variovorax paradoxus B4
复制标题
DOI：
10.1016/j.enzmictec.2018.09.007
复制
发表时间：
2019-01-01
影响因子：
3.4
通讯作者：
Steinbuechel, Alexander
中科院分区：
文献类型：
--
作者：
Brandt, Ulrike;Galant, Gulsina;Steinbuechel, Alexander
硫醇双加氧酶是一种非血红素单核铁蛋白，属于Cupin超家族。2014年，Variovorax paroxus B4的硫代丁二酸双加氧酶(MSDO)被鉴定为另一种细菌半胱氨酸双加氧酶(CDO)同源物，催化硫代丁二酸酯(MS)转化为琥珀酸和亚硫酸盐。为了进一步了解潜在的重要氨基酸残基对酶活性的影响，我们产生并分析了七个酶变异体。(I)三个变异体分别由亮氨酸取代一个保守的组氨酸残基，它们要么被认为是铁(II)辅助因子(H93和H95)的配位所必需的，要么被认为对底物在活性部位内的定位(H163)是重要的。相应的酶变异体完全失活，证实了它们对酶活性的重要作用。(Ii)突变C100S也导致了一种失活的酶，证明了它对蛋白质的稳定性或活性的重要性。(3)对于真核CDO，假设底物定位存在一个氢键网络，相应的氨基酸基本上存在于MSDO中。与野生型的0.06 mM相比，MsdoQ64A突变的K-m增加了0.29 mM，但对比活性没有显著影响。(4)突变体MsdoR66A即使在高量的酶作用下也只表现出很低的活性，这表明该残基可能对催化很重要。(5)突变Y165F的作用不强，其比活力为10.22mU·mol·min~(-1)mg~(-1)蛋白，K-m值为0.06 mM，与野生型酶有很高的相似性。这个残基对应于人类CDO的Y157，它是催化三联体的一部分，被认为参与底物定位。显然，另一个残基可以在Msdo中发挥这一作用，因为Y165的损失没有很强的影响。
Thiol dioxygenases are non-heme mononuclear-iron proteins and belong to the cupin superfamily. In 2014, mercaptosuccinate dioxygenase (Msdo) of Variovorax paradoxus B4 was identified as another bacterial cysteine dioxygenase (Cdo) homolog catalyzing the conversion of mercaptosuccinate (MS) into succinate and sulfite. To gain further insights into potentially important amino acid residues for enzyme activity, seven enzyme variants were generated and analyzed. (i) Three variants comprised the substitution of one conserved histidine residue each by leucine, either supposed to be mandatory for coordination of the Fe(II) cofactor (H93 and H95) or to be important for substrate positioning within the active site (H163). The corresponding enzyme variants were completely inactive confirming their essential roles for enzyme activity. (ii) Mutation C100S resulted as well in an inactive enzyme demonstrating its importance for either stability or activity of the protein. (iii) For eukaryotic Cdo, a hydrogen bond network for substrate positioning was postulated, and the corresponding amino acids are basically present in Msdo. Albeit the MsdoQ64A mutation exhibited an increased K-m of 0.29 mM when compared to the wildtype with 0.06 mM, it did not significantly affect the specific activity. (iv) The variant MsdoR66A showed only very low activity even when high amounts of enzyme were applied indicating that this residue might be important for catalysis. (v) No strong effect had the mutation Y165F for which a specific enzyme activity of 10.22 mu mol min(-1) mg(-1) protein and a K-m value of 0.06 mM with high similarity to those of the wildtype enzyme were obtained. This residue corresponds to Y157 of human Cdo, which is part of the catalytic triad and is supposed to be involved in substrate positioning. Apparently, another residue could fulfill this role in Msdo, since the loss of Y165 did not have a strong effect.