Department of Bioengineering, University of California, Berkeley, California.
Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio.
Protein Sci. 2018 Feb;27(2):402-410. doi: 10.1002/pro.3324. Epub 2017 Nov 28.
The precursor to all tetrapyrroles is 5-aminolevulinic acid, which is made either via the condensation of glycine and succinyl-CoA catalyzed by an ALA synthase (the C4 or Shemin pathway) or by a pathway that uses glutamyl-tRNA as a precursor and involves other enzymes (the C5 pathway). Certain ALA synthases also catalyze the cyclization of ALA-CoA to form 2-amino-3-hydroxycyclopent-2-en-1-one. Organisms with synthases that possess this second activity nevertheless rely upon the C5 pathway to supply ALA for tetrapyrrole biosynthesis. The C N units are components of a variety of secondary metabolites. Here, we show that an ALA synthase used exclusively for tetrapyrrole biosynthesis is also capable of catalyzing the cyclization reaction, albeit at much lower efficiency than the dedicated cyclases. Two absolutely conserved serines present in all known ALA-CoA cyclases are threonines in all known ALA synthases, suggesting they could be important in distinguishing the functions of these enzymes. We found that purified mutant proteins having single and double substitutions of the conserved residues are not improved in their respective alternate activities; rather, they are worse. Protein structural modeling and amino acid sequence alignments were explored within the context of what is known about the reaction mechanisms of these two different types of enzymes to consider what other features are important for the two activities.
所有四吡咯的前体是 5-氨基乙酰丙酸，它可以通过 ALA 合酶（C4 或 Shemin 途径）催化甘氨酸和琥珀酰辅酶 A 的缩合形成，或者通过使用谷氨酰-tRNA 作为前体并涉及其他酶的途径（C5 途径）形成。某些 ALA 合酶还可以催化 ALA-CoA 的环化形成 2-氨基-3-羟基环戊-2-烯-1-酮。具有具有这种第二种活性的合酶的生物仍然依赖 C5 途径来提供 ALA 用于四吡咯生物合成。C N 单位是各种次生代谢物的组成部分。在这里，我们表明，专门用于四吡咯生物合成的 ALA 合酶也能够催化环化反应，尽管效率比专用环化酶低得多。所有已知的 ALA-CoA 环化酶中存在的两个绝对保守的丝氨酸在所有已知的 ALA 合酶中都是苏氨酸，这表明它们可能在区分这些酶的功能方面很重要。我们发现，具有单个和双取代保守残基的纯化突变蛋白在各自的替代活性中没有改善；相反，它们更差。在已知的这两种不同类型酶的反应机制的背景下，探索了蛋白质结构建模和氨基酸序列比对，以考虑对两种活性重要的其他特征。
