Enhancing Human Spermine Synthase Activity by Engineered Mutations

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.

精胺合酶（Spermine synthase, SMS）是一类能够将亚精胺转化为精胺的酶。研究表明，导致野生型SMS氨基酸序列改变的基因缺陷，会引发斯奈德-罗宾逊综合征——这是一种伴随骨质疏松症、面部不对称、体型消瘦、肌张力低下及非特异性运动障碍的轻中度智力障碍疾病。这类致病错义突变经计算机模拟与体外实验证实，可通过破坏SMS二聚体/单体的稳定性，或直接影响其活性位点的氢键网络，进而干扰野生型SMS的正常功能。与既往研究不同，本研究通过跨物种序列信息移植的方式，人工工程化改造得到催化活性更优的SMS变体。经实验验证，携带4处氨基酸取代位点的该变体，其催化活性显著高于野生型SMS。该变体功能增强的机制包括：单体稳定性提升、质子供体催化残基的pKa值降低、SMS分子周围相对于底物的静电势能空间分布得到优化，以及被推测为活性位点入口的裂隙的机械振动频率升高。本研究表明，野生型SMS相对于亚精胺→精胺的催化反应，并未经过特别的进化优化。鉴于目前尚未在健康个体中检测到该基因的非同义单核苷酸多态性（non-synonymous single nucleotide polymorphism, nsSNP），由此可推测，人类SMS的功能被精确调控以维持野生型状态，任何功能偏离均会引发疾病且对机体有害。