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.

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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 (H2O2). 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.