DataSheet1_An Extended C-Terminus, the Possible Culprit for Differential Regulation of 5-Aminolevulinate Synthase Isoforms.pdf

5-Aminolevulinate synthase (ALAS; E.C. 2.3.1.37) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the key regulatory step of porphyrin biosynthesis in metazoa, fungi, and α-proteobacteria. ALAS is evolutionarily related to transaminases and is therefore classified as a fold type I PLP-dependent enzyme. As an enzyme controlling the key committed and rate-determining step of a crucial biochemical pathway ALAS is ideally positioned to be subject to allosteric feedback inhibition. Extensive kinetic and mutational studies demonstrated that the overall enzyme reaction is limited by subtle conformational changes of a hairpin loop gating the active site. These findings, coupled with structural information, facilitated early prediction of allosteric regulation of activity via an extended C-terminal tail unique to eukaryotic forms of the enzyme. This prediction was subsequently supported by the discoveries that mutations in the extended C-terminus of the erythroid ALAS isoform (ALAS2) cause a metabolic disorder known as X-linked protoporphyria not by diminishing activity, but by enhancing it. Furthermore, kinetic, structural, and molecular modeling studies demonstrated that the extended C-terminal tail controls the catalytic rate by modulating conformational flexibility of the active site loop. However, the precise identity of any such molecule remains to be defined. Here we discuss the most plausible allosteric regulators of ALAS activity based on divergences in AlphaFold-predicted ALAS structures and suggest how the mystery of the mechanism whereby the extended C-terminus of mammalian ALASs allosterically controls the rate of porphyrin biosynthesis might be unraveled.

5-氨基卟啉酸合酶（ALAS；EC 2.3.1.37）是一种依赖于吡哆醛-5'-磷酸（PLP）的酶，催化多细胞生物、真菌和α-蛋白菌中卟啉生物合成的关键调控步骤。ALAS在进化上与转氨酶相关，因此被归类为I型折叠PLP依赖性酶。作为控制关键限速步骤的酶，ALAS在关键生化途径中占据着理想的位置，易受变构反馈抑制。广泛的动力学和突变研究显示，整体酶反应受控于调控活性位点的发夹环的微妙构象变化。这些发现，结合结构信息，促进了通过独特的C端延长尾预测变构调控活性的早期研究。这一预测随后得到了发现的支持，即红系ALAS同种型（ALAS2）的C端延长突变会导致一种称为X连锁原卟啉病的代谢紊乱，并非通过降低活性，而是通过增强活性。此外，动力学、结构和分子建模研究表明，C端延长尾通过调节活性位点环的构象灵活性来控制催化速率。然而，此类分子的精确身份尚未确定。在此，我们基于AlphaFold预测的ALAS结构差异，讨论了最可能的ALAS活性变构调节剂，并提出了解开哺乳动物ALASs的C端延长尾如何通过变构调控卟啉生物合成速率之谜的可能途径。