N-methyl Proline Research Articles

Metabolomic Markers of Southern Dietary Patterns in the Jackson Heart Study.

New biomarkers are needed that are representative of dietary intake. We assess metabolites associated with Southern dietary patterns in 1401 Jackson Heart Study participants. Three dietary patterns are empirically derived using principal component analysis: meat and fast food, fish and vegetables, and starchy foods. We randomly select two subsets of the study population: two-third sample for discovery (n = 934) and one-third sample for replication (n = 467). Among the 327 metabolites analyzed, 14 are significantly associated with the meat and fast food dietary pattern, four are significantly associated with the fish and vegetables dietary pattern, and none are associated with the starchy foods dietary pattern in the discovery sample. In the replication sample, nine remain associated with the meat and fast food dietary pattern [indole-3-propanoic acid, C24:0 lysophosphatidylcholine (LPC), N-methyl proline, proline betaine, C34:2 phosphatidylethanolamine (PE) plasmalogen, C36:5 PE plasmalogen, C38:5 PE plasmalogen, cotinine, hydroxyproline] and three remain associated with the fish and vegetables dietary pattern [1,7-dimethyluric acid, C22:6 lysophosphatidylethanolamine, docosahexaenoic acid (DHA)]. Twelve metabolites are discovered and replicated in association with dietary patterns detected in a Southern U.S. African-American population, which could be useful as biomarkers of Southern dietary patterns.

MtpB, a member of the MttB superfamily from the human intestinal acetogen Eubacterium limosum, catalyzes proline betaine demethylation

The trimethylamine methyltransferase MttB is the founding member of a widely distributed superfamily of microbial proteins. Genes encoding most members of the MttB superfamily lack the codon for pyrrolysine that distinguishes previously characterized trimethylamine methyltransferases, leaving the function(s) of most of the enzymes in this superfamily unknown. Here, investigating the MttB family member MtpB from the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that excretes N-methyl proline during growth on proline betaine, we demonstrate that MtpB catalyzes anoxic demethylation of proline betaine. MtpB along with MtqC (a corrinoid protein) and MtqA (a methylcorrinoid:tetrahydrofolate methyltransferase) was much more abundant in E. limosum cells grown on proline betaine than on lactate. We observed that recombinant MtpB methylates Co(I)-MtqC in the presence of proline betaine and that other quaternary amines are much less preferred substrates. MtpB, MtqC, and MtqA catalyze tetrahydrofolate methylation with proline betaine, thereby forming a key intermediate in the Wood-Ljungdahl acetogenesis pathway. To our knowledge, MtpB methylation of Co(I)-MtqC for the subsequent methylation of tetrahydrofolate represents the first described anoxic mechanism of proline betaine demethylation. The activities of MtpB and associated proteins in acetogens or other anaerobes provide a possible mechanism for the production of N-methyl proline by the gut microbiome. MtpB's activity characterized here strengthens the hypothesis that much of the MttB superfamily comprises quaternary amine-dependent methyltransferases.

Re-print of “Theoretical Investigation and Reinterpretation of the Decomposition of Lithiated Proline and N-Methyl Proline”

Lithium cation complexes of proline (Pro) and N-methyl proline (NMP) have been collisionally activated with xenon in a guided ion beam tandem mass spectrometer (GIBMS). In addition to the loss of the intact ligand, Pro and NMP, we observed two prominent fragmentation pathways involving the loss of (CO+LiOH) and (CO+H2O). Quantum chemical calculations at the B3LYP/6-311+G(d, p) level are used to explore the reaction mechanisms of these Li+(Pro) and Li+(NMP) fragmentations. Complete potential energy surfaces including all intermediates and transition states are elucidated for the two fragmentation processes in both systems. Theoretical molecular parameters for the rate-limiting transition states are then used to analyze the experimental data. The experimental threshold energies are compared with single point energies calculated at six different levels of theory. Reasonable agreement between experiment and some levels of theory indicate that loss of the intact amino acid competes with the loss of CO over a tight transition state in both Li+(Pro) and Li+(NMP) systems. Once CO is lost, efficient loss of H2O can occur at lower or comparable energy and is followed at somewhat higher energies by loss of LiOH, both proceeding by loose transition states. Overall, we find that MP2(full)/6-311+G(2d, 2p) gives the best agreement with the experimental threshold energies and the qualitative characteristics of the competing reactions. This study refines the bond energy of Li+ to Pro by considering competition with CO loss and lowers the value previously published by 24±12kJ/mol, with methylation of proline increasing the bond energy to Li+ by 9±15kJ/mol.

Theoretical investigation and reinterpretation of the decomposition of lithiated proline and N-methyl proline

Lithium cation complexes of proline (Pro) and N-methyl proline (NMP) have been collisionally activated with xenon in a guided ion beam tandem mass spectrometer (GIBMS). In addition to the loss of the intact ligand, Pro and NMP, we observed two prominent fragmentation pathways involving the loss of (CO+LiOH) and (CO+H2O). Quantum chemical calculations at the B3LYP/6-311+G(d, p) level are used to explore the reaction mechanisms of these Li+(Pro) and Li+(NMP) fragmentations. Complete potential energy surfaces including all intermediates and transition states are elucidated for the two fragmentation processes in both systems. Theoretical molecular parameters for the rate-limiting transition states are then used to analyze the experimental data. The experimental threshold energies are compared with single point energies calculated at six different levels of theory. Reasonable agreement between experiment and some levels of theory indicate that loss of the intact amino acid competes with the loss of CO over a tight transition state in both Li+(Pro) and Li+(NMP) systems. Once CO is lost, efficient loss of H2O can occur at lower or comparable energy and is followed at somewhat higher energies by loss of LiOH, both proceeding by loose transition states. Overall, we find that MP2(full)/6-311+G(2d, 2p) gives the best agreement with the experimental threshold energies and the qualitative characteristics of the competing reactions. This study refines the bond energy of Li+ to Pro by considering competition with CO loss and lowers the value previously published by 24±12kJ/mol, with methylation of proline increasing the bond energy to Li+ by 9±15kJ/mol.

Role of methylation on the thermochemistry of alkali metal cation complexes of amino acids: N-methyl proline

Quantitative thermodynamic information is obtained from the study of the gas-phase interactions of the alkali metal cation complexes of N-methyl proline (NMP) with Xe using a guided ion beam tandem mass spectrometer (GIBMS). Absolute bond dissociation energies (BDEs) of M+=Li+, Na+, K+, and Rb+ to NMP are determined experimentally from threshold collision-induced dissociation (TCID) measurements of the M+(NMP) complexes. Analysis of their kinetic energy cross sections provide the 0K bond enthalpies after accounting for unimolecular decay rates, internal energy of reactant ions, and multiple ion-molecule collisions. Quantum chemical calculations of the M+(NMP) BDEs are found to be in good agreement with the experimental values, establishing that the zwitterionic form is the lowest energy structure for all the metal ion complexes. Compared to M+(Pro) BDEs, the metal binding in these zwitterions is slightly enhanced by the CH3 group on the ring nitrogen, presumably a result of an inductive effect and its higher polarizability. More profound consequences of the methyl group emerge in the charge-solvated conformers calculated for M+(NMP) where it directs multiple conformations of the pyrrolidine ring. This is unlike the ring puckering phenomenon seen in M+(Pro) complexes, where fewer conformations are found, apparently because inversion at the nitrogen center is more facile.

Systematic Study of the Structures of Potassiated Tertiary Amino Acids: Salt Bridge Structures Dominate

The gas-phase structures of a series of potassiated tertiary amino acids have been systematically investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser, ion mobility spectrometry (IMS), and computational modeling. The examined analytes comprise a set of five linear N,N-dimethyl amino acids derived from N,N-dimethyl glycine and three cyclic N-methyl amino acids including N-methyl proline. The number of methylene groups in either the alkyl chain of the linear members or in the ring of the cyclic members of the series is gradually varied. The spectra of the cyclic potassiated molecular ions are similar and well resolved, whereas the clear signals in the respective spectra of the linear analytes increasingly overlap with longer alkyl chains. Measured IRMPD spectra are compared to spectra calculated at the B3LYP/6-311++G(2d,2p) level of theory to identify the structures present in the experimental studies. On the basis of these experiments and calculations, all potassiated molecular ions of this series adopt salt bridge structures in the gas phase, involving bidentate coordination of the potassium cation to the carboxylate moiety. The assigned salt bridge structures are predicted to be the global minima on the potential energy surfaces. IMS cross-section measurements of the potassiated systems show a monotonic increase with growing system size, suggesting that the precursor ions adopt the same type of structure and comparisons between experimental and theoretical cross sections are consistent with salt bridge structures and the IRMPD results.

Kinetic Determination of Potassium Affinities by IRMPD: Elucidation of Precursor Ion Structures

Dissociation kinetics of the K(+) loss reaction of three potassiated tertiary amino acids (Scheme 1) were studied by infrared multiple photon dissociation (IRMPD) in a Fourier transform ion cyclotron resonance (FT ICR)-MS instrument. The aim of the study was to probe if a kinetic study by IRMPD can yield useful information on the ion structure of the precursor ion species. The measured activation energy values determined by IRMPD are related to the potassium affinity, DeltaH(K(+)), of N-methyl proline determined by threshold collision-induced dissociation experiments. By appropriate scaling with this reference value, the experimentally determined activation energy values for the K(+) loss are transformed into respective potassium affinities, DeltaH(K(+))(IRMPD). These values match the calculated potassium affinity values for salt bridge (SB) structures, DeltaH(K(+))(SB), substantially better than those for canonical structures with a single formal charge site (charge solvation (CS)), thereby allowing structure identification. This conclusion is consistent with other spectroscopic data, which yielded unambiguous evidence of these tertiary amino acids adopting SB structures in the gas phase. This study demonstrates that IRMPD can be applied to determine individual ion structures in the gas phase, given that adequate reference values are available for proper scaling.

The stachydrine catabolism region in Sinorhizobium meliloti encodes a multi-enzyme complex similar to the xenobiotic degrading systems in other bacteria

Stachydrine (proline betaine) can be used by Sinorhizobium meliloti as a source of carbon and nitrogen. Catabolism depends on an initial N-demethylation, after which the resultant N-methyl proline enters general metabolism. Deletion and insertion mutagenesis demonstrated that the information necessary for catabolism is carried on the symbiotic plasmid (pSym) distal to nodD2 and the nod-nif cluster. Sequencing of an 8.5kb fragment spanning this region revealed four open reading frames with functional homology to known proteins, including a putative monooxygenase and a putative NADPH-FMN-reductase, which were shown by insertional and frame-shift mutagenesis to be necessary for stachydrine catabolism. Other open reading frames, encoding a putative flavoprotein and a repressor, were judged not to be required for stachydrine catabolism, since they were not included in a fragment capable of complementing a deletion of the entire stc region. Sequence and mutagenesis data suggest that stachydrine is demethylated by an iron-sulfur monooxygenase of the Rieske type with a requirement for a specific reductase. The stc catabolic cluster, therefore, resembles xenobiotic degradation in other bacteria and recalls rhizopine catabolism in S. meliloti. Stachydrine appears to have multiple roles in osmoprotection, nutrition and nodulation. Genes involved in stachydrine catabolism are also necessary for carnitine degradation; thus, they could be important in the catabolism of a variety of root exudates and mediate other relationships.