TY - JOUR

T1 - Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer

AU - Quareshy, Mussa

AU - Shanmugam, Muralidharan

AU - Townsend, Eleanor

AU - Jameson, Eleanor

AU - Bugg, Timothy D H

AU - Cameron, Alexander D

AU - Chen, Yin

N1 - Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

PY - 2021/1/23

Y1 - 2021/1/23

N2 - Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail α3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located >40 Å apart in the same monomer but adjacent in two neighboring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in intersubunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.

AB - Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail α3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located >40 Å apart in the same monomer but adjacent in two neighboring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in intersubunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.

KW - Carnitine/metabolism

KW - Catalysis

KW - Electron Spin Resonance Spectroscopy

KW - Electron Transport

KW - Mixed Function Oxygenases/antagonists & inhibitors

KW - Protein Conformation

KW - Substrate Specificity

U2 - 10.1074/jbc.RA120.016019

DO - 10.1074/jbc.RA120.016019

M3 - Article

C2 - 33158989

VL - 296

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

M1 - 100038

ER -