Quantum Chemistry Calculations for Metabolomics.

A primary goal of metabolomics studies is to fully characterize the small-molecule composition of complex biological and environmental samples. However, despite advances in analytical technologies over the past two decades, the majority of small molecules in complex samples are not readily identifiable due to the immense structural and chemical diversity present within the metabolome. Current gold-standard identification methods rely on reference libraries built using authentic chemical materials ("standards"), which are not available for most molecules. Computational quantum chemistry methods, which can be used to calculate chemical properties that are then measured by analytical platforms, offer an alternative route for building reference libraries, i.e., in silico libraries for "standards-free" identification. In this review, we cover the major roadblocks currently facing metabolomics and discuss applications where quantum chemistry calculations offer a solution. Several successful examples for nuclear magnetic resonance spectroscopy, ion mobility spectrometry, infrared spectroscopy, and mass spectrometry methods are reviewed. Finally, we consider current best practices, sources of error, and provide an outlook for quantum chemistry calculations in metabolomics studies. We expect this review will inspire researchers in the field of small-molecule identification to accelerate adoption of in silico methods for generation of reference libraries and to add quantum chemistry calculations as another tool at their disposal to characterize complex samples.

Evaluating the Accuracy of the QCEIMS Approach for Computational Prediction of Electron Ionization Mass Spectra of Purines and Pyrimidines.

Mass spectrometry is the most commonly used method for compound annotation in metabolomics. However, most mass spectra in untargeted assays cannot be annotated with specific compound structures because reference mass spectral libraries are far smaller than the complement of known molecules. Theoretically predicted mass spectra might be used as a substitute for experimental spectra especially for compounds that are not commercially available. For example, the Quantum Chemistry Electron Ionization Mass Spectra (QCEIMS) method can predict 70 eV electron ionization mass spectra from any given input molecular structure. In this work, we investigated the accuracy of QCEIMS predictions of electron ionization (EI) mass spectra for 80 purine and pyrimidine derivatives in comparison to experimental data in the NIST 17 database. Similarity scores between every pair of predicted and experimental spectra revealed that 45% of the compounds were found as the correct top hit when QCEIMS predicted spectra were matched against the NIST17 library of >267,000 EI spectra, and 74% of the compounds were found within the top 10 hits. We then investigated the impact of matching, missing, and additional fragment ions in predicted EI mass spectra versus ion abundances in MS similarity scores. We further include detailed studies of fragmentation pathways such as retro Diels-Alder reactions to predict neutral losses of (iso)cyanic acid, hydrogen cyanide, or cyanamide in the mass spectra of purines and pyrimidines. We describe how trends in prediction accuracy correlate with the chemistry of the input compounds to better understand how mechanisms of QCEIMS predictions could be improved in future developments. We conclude that QCEIMS is useful for generating large-scale predicted mass spectral libraries for identification of compounds that are absent from experimental libraries and that are not commercially available.

Dimerization misalignment in human glutamate-oxaloacetate transaminase variants is the primary factor for PLP release.

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), plays an essential role in the catalytic mechanism of various proteins, including human glutamate-oxaloacetate transaminase (hGOT1), an important enzyme in amino acid metabolism. A recent molecular and genetic study showed that the E266K, R267H, and P300L substitutions in aspartate aminotransferase, the Arabidopsis analog of hGOT1, genetically suppress a developmentally arrested Arabidopsis RUS mutant. Furthermore, CD analyses suggested that the variants exist as apo proteins and implicated a possible role of PLP in the regulation of PLP homeostasis and metabolic pathways. In this work, we assessed the stability of PLP bound to hGOT1 for the three variant and wildtype (WT) proteins using a combined 6 µs of molecular dynamics (MD) simulation. For the variants and WT in the holo form, the MD simulations reproduced the "closed-open" transition needed for substrate binding. This conformational transition was associated with the rearrangement of the P15-R32 small domain loop providing substrate access to the R387/R293 binding motif. We also showed that formation of the dimer interface is essential for PLP affinity to the active site. The position of PLP in the WT binding site was stabilized by a unique hydrogen bond network of the phosphate binding cup, which placed the cofactor for formation of the covalent Schiff base linkage with K259 for catalysis. The amino acid substitutions at positions 266, 267, and 300 reduced the structural correlation between PLP and the protein active site and/or integrity of the dimer interface. Principal component analysis and energy decomposition clearly suggested dimer misalignment and dissociation for the three variants tested in our work. The low affinity of PLP in the hGOT1 variants observed in our computational work provided structural rationale for the possible role of vitamin B6 in regulating metabolic pathways.

Structure of the Bacillus anthracis dTDP-L-rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA).

L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), from Bacillus anthracis was determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Šresolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs. However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.