Enhancing human spermine synthase activity by engineered mutations.
Enhancing human spermine synthase activity by engineered mutations.
复制标题
通过工程突变增强人的精子合酶活性。
DOI：
10.1371/journal.pcbi.1002924
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发表时间：
2013
影响因子：
4.3
通讯作者：
Alexov E
中科院分区：
文献类型：
--
作者：
Zhang Z;Zheng Y;Petukh M;Pegg A;Ikeguchi Y;Alexov E
精胺合成酶（SMS）是一种将精脒转化为精胺的酶。研究表明，导致野生型SMS氨基酸变化的基因缺陷导致Snyder-Robinson综合征，这是一种与骨质疏松症、面部不对称、瘦体质、张力减退和非特异性运动障碍相关的轻度至中度精神残疾。这些致病错义突变在计算机模拟和体外均被证明通过使SMS二聚体/单体不稳定或直接影响SMS活性位点的氢键网络来影响SMS的野生型功能。与这些研究相反，在这里，我们报告了一种更有效的SMS变体的人工工程，通过从另一种生物体转移序列信息。实验证实，携带四个氨基酸取代的变体比野生型催化活性更高。增加的功能性是由于增强的单体稳定性，降低质子供体催化残基的pKa，优化的空间分布的SMS周围的静电势相对于基板，和增加的频率的裂缝被推定为朝向活性位点的门的机械振动。该研究表明，野生型SMS在亚精胺→精胺的反应方面没有特别进化优化。考虑到目前在健康个体中没有检测到变异（非同义单核苷酸多态性，nsSNP），可以推测人类SMS功能被精确地调整到其野生型，并且任何偏离都是不需要的和致病的。蛋白质不断受到进化的压力，以确保生物体的生存和繁殖。与此同时，蛋白质的氨基酸序列发生突变，其中一些可能导致疾病，而另一些可能反映了人群中的自然差异（非同义单核苷酸多态性，nsSNP）。在这项研究中，我们研究了人类精胺合酶（HsSMS），目前还没有nsSNPs，而罕见的疾病突变已知会导致斯奈德-罗宾逊综合征。这种蛋白质有什么特别之处？也许HsSMS对其功能进行了很好的优化，以至于野生型序列的任何变化都应该降低其性能。为了验证这种可能性，我们设计了一种HsSMS突变体，具有增强的稳定性、静电和机械性能。实验证实突变体是比野生型更好的酶。因此，HsSMS在其酶反应方面并未进行进化优化，其氨基酸序列仅在患病个体中存在差异，而到目前为止，发现其序列在所有健康个体中是相同的。因此，可以推测，HsSMS功能被精确地调整为野生型特征，因此任何偏离都是不需要的并且是致病的。
Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing. Proteins are constantly subjected to evolutionary pressure to assure the organism's survival and reproduction. At the same time, the proteins' amino acid sequence undergoes mutations, some of which may cause diseases while others may be reflecting natural differences within the population (non-synonymous single nucleotide polymorphism, nsSNP). In this study we examine the human spermine synthase (HsSMS), for which currently there are no nsSNPs, while rare disease mutations are known to cause Snyder-Robinson syndrome. What is so special with this protein? Maybe the HsSMS is so well optimized for its function that any change of the wild type sequence should be degrading its performance. To check such a possibility, we engineered a mutant of HsSMS with enhanced stability, electrostatic and mechanical properties. The mutant was confirmed experimentally to be a better enzyme than the wild type. Thus, the HsSMS is not evolutionally optimized with respect to its enzymatic reaction, its amino acid sequence differs only in sick individuals and so far its sequence was found to be identical in all healthy individuals. Therefore, it can be speculated that the HsSMS function is precisely tuned toward the wild type characteristics such so any deviation is unwanted and is disease-causing.
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