One ring closer to a closure: the crystal structure of the ES3 hydroxymethylbilane synthase intermediate,The FEBS Journal

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One ring closer to a closure: the crystal structure of the ES3 hydroxymethylbilane synthase intermediate
The FEBS Journal ( IF 5.5 ) Pub Date : 2023-10-20 , DOI: 10.1111/febs.16982
Helene J. Bustad ₁ , Marthe S. Christie ₁ , Mikko Laitaoja ₂ , Aasne K. Aarsand _{1,

3} , Aurora Martinez ₄ , Janne Jänis ₂ , Juha P. Kallio ₄

Affiliation

Hydroxymethylbilane synthase (HMBS), involved in haem biosynthesis, catalyses the head-to-tail coupling of four porphobilinogens (PBGs) via a dipyrromethane (DPM) cofactor. DPM is composed of two PBGs, and a hexapyrrole is built before the tetrapyrrolic 1-hydroxymethylbilane product is released. During this elongation, stable enzyme (E) intermediates are formed from the holoenzyme, with additional PBG substrates (S): ES, ES₂, ES₃ and ES₄. Native PAGE and mass spectrometry of the acute intermittent porphyria (AIP)-associated HMBS variant p.Arg167Gln demonstrated an increased amount of ES₃. Kinetic parameters indicated catalytic dysfunction, however, the product release was not entirely prevented. Isolation and crystal structure analysis of the ES₃ intermediate (PDB: 8PND) showed that a pentapyrrole was fully retained within the active site, revealing that polypyrrole elongation proceeds within the active site via a third interaction site, intermediate pyrrole site 3 (IPS3). The AIP-associated HMBS variant p.Arg195Cys, located on the opposite side to p.Arg167Gln in the active site, accumulated the ES₄ intermediate in the presence of excess PBG, implying that product hydrolysis was obstructed. Arg167 is thus involved in all elongation steps and is a determinant for the rate of enzyme catalysis, whereas Arg195 is important for releasing the product. Moreover, by substituting residues in the vicinity of IPS3, our results indicate that a fully retained hexapyrrole could be hydrolysed in a novel site in proximity of the IPS3.

中文翻译：

一个接近闭合的环：ES3 羟甲基双烷合酶中间体的晶体结构

羟甲基胆烷合酶 (HMBS) 参与血红素生物合成，通过二吡咯甲烷 (DPM) 辅因子催化四种胆色素原 (PBG) 的头尾偶联。 DPM由两个PBG组成，在四吡咯1-羟甲基联烷产物释放之前先构建了一个六吡咯。在此延伸过程中，由全酶形成稳定的酶 (E) 中间体，并具有额外的 PBG 底物 (S)：ES、ES ₂、ES ₃和 ES ₄。急性间歇性卟啉症 (AIP) 相关的 HMBS 变体 p.Arg167Gln 的非变性 PAGE 和质谱分析表明 ES ₃的量增加。动力学参数表明催化功能障碍，然而，产物释放并未完全被阻止。 ES ₃中间体 (PDB: 8PND) 的分离和晶体结构分析表明，五吡咯完全保留在活性位点内，表明聚吡咯在活性位点内通过第三个相互作用位点（中间体吡咯位点 3 (IPS3)）进行延伸。 AIP 相关的 HMBS 变体 p.Arg195Cys 位于活性位点 p.Arg167Gln 的相对侧，在过量 PBG 存在的情况下积累 ES ₄中间体，这意味着产物水解受到阻碍。因此，Arg167 参与所有延伸步骤，并且是酶催化速率的决定因素，而 Arg195 对于释放产物很重要。此外，通过取代 IPS3 附近的残基，我们的结果表明完全保留的六吡咯可以在 IPS3 附近的新位点水解。

更新日期：2023-10-20

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