Carnitine metabolism in the human gut: characterization of the two-component carnitine monooxygenase CntAB from Acinetobacter baumannii

Abstract

Bacterial formation of trimethylamine (TMA) from carnitine in the gut microbiome has been linked to cardiovascular disease. During this process the two-component carnitine monooxygenase (CntAB) catalyzes the oxygen-dependent cleavage of carnitine to TMA and malic semialdehyde. Individual redox states of the reductase CntB and the catalytic component CntA were investigated on the basis of mutagenesis and electron paramagnetic resonance spectroscopic (EPR) approaches. Protein ligands of the flavin mononucleotide (FMN) and the plant-type [2Fe-2S] cluster of CntB and also of the Rieske-type [2Fe-2S] cluster and the mononuclear [Fe] center of CntA were identified. EPR spectroscopy of variant CntA proteins suggested a hierarchical metallocenter maturation, Rieske [2Fe-2S] followed by the mononuclear [Fe] center. NADH-dependent electron transfer via the redox components of CntB and within the trimeric CntA complex for the activation of molecular oxygen was investigated. EPR experiments indicated that the two electrons from NADH were allocated to the plant-type [2Fe-2S] cluster and to FMN in form of a flavin semiquinone radical. Single turnover experiments of this reduced CntB species indicated the translocation of the first electron onto the [Fe] center and the second electron onto the Rieske-type [2Fe-2S] cluster of CntA to finally allow for oxygen activation as basis for carnitine cleavage. EPR spectroscopic investigation of CntA variants indicated an unusual intermolecular electron transfer between the subunits of the CntA trimer via the “bridging” residue Glu-205. On the basis of these data, a redox catalytic cycle for carnitine monooxygenase was proposed.

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

“…This is consistent with predominant (>95 %) oxidized state for the Rieske center and the monoFe as a ferrous oxidation state (Figure S7; wide‐swept, cw‐EPR, top panel), as reported for other Rieske‐type oxygenases [12, 13] . This is in contrast to a recent report, where a high‐spin, S =5/2, ferric oxidation state is observed for Ab CntA [36] . It is unclear whether this is due to the autooxidation of the non‐heme iron center.…”

“…[12,13] This is in contrast to a recent report, where a high-spin, S = 5/2, ferric oxidation state is observed for AbCntA. [36] It is unclear whether this is due to the autooxidation of the non-heme iron center. The observa- Figure 1.…”

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

“…2 D ). This was also independently validated by a recent study showing that both E205D and E205Q mutants lost enzyme activity and the ability for electron transfer to the active center ( 13 , 50 ). Binding of the substrate to the catalytic mononuclear Fe center caused a shift in g 3 tensor from 3810 G to 3750 G ( Fig.…”

“…Rieske oxygenases are challenging proteins to study, and this was no different in the case of CntA. Different expression strategies or the choice of buffers and additives may have contributed to the apparent differences in enzyme activities among studies of CntA protein ( 13 , 50 ). Owing to sensitivity to oxygen, considerable effort was made in our work in order to obtain CntA crystals.…”

The MttB superfamily member MtyB from the human gut symbiont Eubacterium limosum is a cobalamin-dependent γ-butyrobetaine methyltransferase

“…A direct route to TMA production from choline is afforded by CutC, the choline-TMA lyase ( 29 , 30 , 31 ). TMA can also be directly produced from l -carnitine as mediated by CntAB, the l -carnitine monooxygenase ( 32 , 33 ). However, most of the TMA produced in the gut from l -carnitine is made with the intermediacy of γ-butyrobetaine ( 15 ) ( Fig.…”