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

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 based on mutagenesis and electron paramagnetic resonance (EPR) spectroscopic 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 the 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 a 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.

A phosphatidic acid-binding protein is important for lipid homeostasis and adaptation to anaerobic biofilm conditions in Pseudomonas aeruginosa.

A quantitative Pseudomonas aeruginosa proteomics approach revealed increased abundance of the so-far uncharacterized protein PA3911 in anaerobic biofilms grown under conditions of the cystic fibrosis lung. Physiological relevance of ORF PA3911 was demonstrated, inter alia, using phenotype microarray experiments. The mutant strain showed increased susceptibility in the presence of antimicrobials (minocycline, nafcillin, oxacillin, chloramphenicol and thiamphenicol), enhanced twitching motility and significantly impaired biofilm formation. PA3911 is a soluble, cytoplasmic protein in P. aeruginosa In protein-lipid overlay experiments, purified PA3911 bound specifically to phosphatidic acid (PA), the central hub of phospholipid metabolism. Structure-guided site-directed mutagenesis was used to explore the proposed ligand-binding cavity of PA3911. Protein variants of Leu56, Leu58, Val69 and Leu114 were shown to impair PA interaction. A comparative shotgun lipidomics approach demonstrated a multifaceted response of P. aeruginosa to anaerobic conditions at the lipid head group and fatty acid level. Lipid homeostasis in the PA3911 mutant strain was imbalanced with respect to lysophosphatidylcholine, phosphatidylcholine and diacylglycerol under anaerobic and/or aerobic conditions. The impact of the newly identified PA-binding protein on lipid homeostasis and the related macroscopic phenotypes of P. aeruginosa are discussed.