Singapore Institute of Food and Biotechnology Innovation (SIFBI), Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore.
Toulouse Biotechnolgy Institute, Université de Toulouse, CNRS, INRAE, INSA, 31077 Toulouse, France.
Biomolecules. 2023 Feb 15;13(2):366. doi: 10.3390/biom13020366.
Molecular biodiversity results from branched metabolic pathways driven by enzymatic regioselectivities. An additional complexity occurs in metabolites with an internal structural symmetry, offering identical extremities to the enzymes. For example, in the terpene family, β-carotene presents two identical terminal closed-ring structures. Theses cycles can be hydroxylated by cytochrome P450s from the CYP97 family. Two sequential hydroxylations lead first to the formation of monohydroxylated β-cryptoxanthin and subsequently to that of dihydroxylated zeaxanthin. Among the CYP97 dihydroxylases, CYP97H1 from has been described as the only monohydroxylase. This study aims to determine which enzymatic domains are involved in this regioselectivity, conferring unique monohydroxylase activity on a substrate offering two identical sites for hydroxylation. We explored the effect of truncations, substitutions and domain swapping with other CYP97 members and found that CYP97H1 harbours a unique N-terminal globular domain. This CYP97H1 N-terminal domain harbours a hydrophobic patch at the entrance of the substrate channel, which is involved in the monohydroxylase activity of CYP97H1. This domain, at the surface of the enzyme, highlights the role of distal and non-catalytic domains in regulating enzyme specificity.
分子生物多样性源于分支代谢途径，这些途径由酶的区域选择性驱动。在具有内部结构对称性的代谢物中会出现额外的复杂性，为酶提供相同的末端。例如，在萜烯家族中，β-胡萝卜素呈现出两个相同的末端闭环结构。这些环可以被 CYP97 家族的细胞色素 P450 羟化。两次连续的羟化首先导致单羟基化β-隐黄质的形成，随后是二羟基化玉米黄质的形成。在 CYP97 二羟化酶中， 中的 CYP97H1 被描述为唯一的单羟化酶。本研究旨在确定哪些酶结构域参与这种区域选择性，赋予具有两个相同羟基化位点的底物独特的单羟化酶活性。我们探索了截断、取代和与其他 CYP97 成员的结构域交换的影响，发现 CYP97H1 具有独特的 N 端球状结构域。CYP97H1 的 N 端结构域在底物通道的入口处具有疏水性斑块，该斑块参与 CYP97H1 的单羟化酶活性。这个位于酶表面的结构域突出了远端和非催化结构域在调节酶特异性方面的作用。
