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

Abstract

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 a3 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 neighbouring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in inter-subunit 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.

Light‐Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske‐Type Oxygenase from Human Microbiota

“… Schematic of NADH photoactivation coupled with EPR used to monitor the electron transfer from NADH to the Rieske‐type, [2Fe‐2S] 2+ in Ab CntA variants (e.g. Ab CntA; PDB ID: 6Y8J) [9b] and subsequent, carnitine catalysis takes place at the monoFe (where EPR‐silent, ferrous center is converted into high‐spin, ferric species; S =5/2) when the photo‐reduced, Ab CntA‐WT sample was annealed at higher temperatures (more details; see Figure 4). …”

“…[4][5][6] Several quaternary ammonium oxygenases have been identified in the last decade, and studies of their active-sites have been reported. [7][8][9] These enzymes comprise a reductase protein containing ferredoxin domain or Rieske [2Fe-2S] 2+ cluster, and an oxygenase protein containing a non-heme, mononuclear iron center (monoFe). [8,10] The carnitine oxygenase (CntA) and reductase (CntB) from Acinetobacter baumannii convert carnitine into TMA and malic-semi-aldehyde (MSA).…”

“…[8,10] The carnitine oxygenase (CntA) and reductase (CntB) from Acinetobacter baumannii convert carnitine into TMA and malic-semi-aldehyde (MSA). [9] The NADH reductase domain (AbCntB) contains a flavin mononucleotide (FMN) binding site and a plant-type ferredoxin, [2Fe-2S] 2+ cluster. [1,2,9,[11][12][13] The oxygenase domain (AbCntA-WT) is an a 3 homo-trimer and contains the monoFe site and a Rieske-type, [2Fe-2S] 2+ cluster in each asubunit with a head-to-tail interaction between each monomer.…”

“…[9] The NADH reductase domain (AbCntB) contains a flavin mononucleotide (FMN) binding site and a plant-type ferredoxin, [2Fe-2S] 2+ cluster. [1,2,9,[11][12][13] The oxygenase domain (AbCntA-WT) is an a 3 homo-trimer and contains the monoFe site and a Rieske-type, [2Fe-2S] 2+ cluster in each asubunit with a head-to-tail interaction between each monomer. [11] The recent crystal structure of AbCntA with (PDB code: 6Y8J) [9B] and without carnitine (PDB code: 6Y9D) [9B] shows that these two redox centers are separated by 44 within a a-subunit, so it is proposed that the electron transfer occurs across the neighboring subunits separated by % 12 (Figure 1), via an electron transfer pathway containing bridging glutamate E205.…”

“…[9B] The mutation of E205 (E205A, E205Q and E205D) causes loss of catalytic activity. [9] Previous bio-physical studies on the Rieske-type oxygenases demonstrated that reductase domain mediates electron-transfer between the reductase and oxygenase domains, and the subsequent catalysis takes place at the monoFe. [11][12][13] The conversion of carnitine into TMA and MSA involves two single-electron transfer processes from AbCntB to AbCntA.…”