Department of Structural Biology of Prokaryotic Organisms, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.
Laboratory of Protein Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.
Acta Crystallogr D Struct Biol. 2022 Jul 1;78(Pt 7):865-882. doi: 10.1107/S2059798322005605. Epub 2022 Jun 14.
The mesophilic cyanobacterium Synechocystis sp. PCC 6803 encodes an S-adenosyl-L-homocysteine hydrolase (SAHase) of archaeal origin in its genome. SAHases are essential enzymes involved in the regulation of cellular S-adenosyl-L-methionine (SAM)-dependent methylation reactions. They are usually active as homotetramers or, less commonly, as homodimers. A SAHase subunit is composed of two major domains: a cofactor (NAD)-binding domain and a substrate (S-adenosyl-L-homocysteine)-binding domain. These are connected by a hinge element that is also a coordination site for an alkali-metal cation that influences domain movement during the catalytic cycle. Typically, the highest activity and strongest substrate binding of bacterial SAHases are observed in the presence of K ions. The SAHase from Synechocystis (SynSAHase) is an exception in this respect. Enzymatic and isothermal titration calorimetry studies demonstrated that in contrast to K-dependent SAHases, the activity and ligand binding of SynSAHase are not affected by the presence of any particular alkali ion. Moreover, in contrast to other SAHases, the cyanobacterial enzyme is in an equilibrium of two distinct oligomeric states corresponding to its dimeric and tetrameric forms in solution. To explain these phenomena, crystal structures of SynSAHase were determined for the enzyme crystallized in the presence of adenosine (a reaction byproduct or substrate) and sodium or rubidium cations. The structural data confirm that while SynSAHase shares common structural features with other SAHases, no alkali metal is coordinated by the cyanobacterial enzyme as a result of a different organization of the macromolecular environment of the site that is normally supposed to coordinate the metal cation. This inspired the generation of SynSAHase mutants that bind alkali-metal cations analogously to K-dependent SAHases, as confirmed by crystallographic studies. Structural comparisons of the crystal structure of SynSAHase with other experimental models of SAHases suggest a possible explanation for the occurrence of the cyanobacterial enzyme in the tetrameric state. On the other hand, the reason for the existence of SynSAHase in the dimeric state in solution remains elusive.
嗜热蓝藻集胞藻 PCC 6803 的基因组中编码一个古菌来源的 S-腺苷-L-同型半胱氨酸水解酶（SAHase）。SAHases 是参与细胞 S-腺苷-L-甲硫氨酸（SAM）依赖性甲基化反应调控的必需酶。它们通常作为同源四聚体或较少情况下作为同二聚体发挥活性。SAHase 亚基由两个主要结构域组成：辅因子（NAD）结合结构域和底物（S-腺苷-L-同型半胱氨酸）结合结构域。这两个结构域通过一个铰链元件连接，该铰链元件也是一个碱金属阳离子的配位位点，该阳离子在催化循环中影响结构域运动。通常，在存在 K 离子的情况下，细菌 SAHases 的最高活性和最强底物结合可被观察到。然而，集胞藻的 SAHase（SynSAHase）在这方面是一个例外。酶学和等温滴定量热法研究表明，与依赖 K 的 SAHases 不同，SynSAHase 的活性和配体结合不受任何特定碱金属离子的存在的影响。此外，与其他 SAHases 不同，蓝藻酶处于与其在溶液中的二聚体和四聚体形式相对应的两个不同寡聚状态的平衡中。为了解释这些现象，对在腺苷（反应副产物或底物）和钠离子或铷离子存在下结晶的 SynSAHase 酶进行了晶体结构测定。结构数据证实，虽然 SynSAHase 与其他 SAHases 具有共同的结构特征，但由于金属阳离子配位位点的大分子环境的组织方式不同，蓝藻酶没有配位任何碱金属离子。这启发了生成 SynSAHase 突变体，这些突变体类似地结合碱金属阳离子，正如晶体学研究所证实的那样。SynSAHase 的晶体结构与其他 SAHase 的实验模型的结构比较为蓝藻酶以四聚体状态存在提供了一个可能的解释。另一方面，SynSAHase 在溶液中以二聚体状态存在的原因仍然难以捉摸。
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