Mechanistic insights into allosteric regulation of the reductase component of p -hydroxyphenylacetate 3-hydroxylase by p -hydroxyphenylacetate: a model for effector-controlled activity of redox enzymes

• Published in: RSC chemical biology Vol. 6; no. 1; pp. 81 - 93
• Main Authors: Visitsatthawong, Surawit, Anuwan, Piyanuch, Lawan, Narin, Chaiyen, Pimchai, Wongnate, Thanyaporn
• Format: Journal Article
• Language: English
• Published: England RSC 02.01.2025
• Subjects: Chemistry
• DOI: 10.1039/D4CB00213J
• ISSN: 2633-0679

Understanding how an enzyme regulates its function through substrate or allosteric regulation is crucial for controlling metabolic pathways. Some flavin-dependent monooxygenases (FDMOs) have evolved an allosteric mechanism to produce reduced flavin while minimizing the use of NADH and the production of harmful hydrogen peroxide (H 2 O 2 ). In this work, we investigated in-depth mechanisms of how the reductase component (C1) of p -hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) from Acinetobacter baumanii is allosterically controlled by the binding of HPA, which is a substrate of its monooxygenase counterpart (C2). The C1 structure can be divided into three regions: the N-terminal domain (flavin reductase); a flexible loop; and the C-terminal domain, which is homologous to NadR, a repressor protein having HPA as an effector. The binding of HPA to NadR induces a conformational change in the recognition helix, causing it to disengage from the NadA gene. The HPA binding site of C1 is located at the C-terminal domain, which can be divided into five helices. Molecular dynamics simulations performed on HPA-docked C1 elucidated the allosteric mechanism. The carboxylate group of HPA maintains the salt bridge between helix 2 and the flexible loop. This maintenance shortens the loop between helices 2 and 3, causing helix 3 to disengage from the N-terminal domain. The aromatic ring of HPA induces a conformational change in helices 1 and 5, pulling helix 4, analogous to the recognition helix in NadR, away from the N-terminal domain. A Y189A mutation, obtained from site-saturation mutagenesis, confirms that HPA with an unsuitable conformation cannot induce the conformational change of C1. Additionally, an HPA-independent effect is observed, in which Arg20, an NADH binding residue on the N-terminal domain, occasionally disengages from helix 4. This model provides valuable insights into the allosteric regulation of two-component FDMOs and aromatic effector systems.