Millisecond mix-and-quench crystallography (MMQX) enables time-resolved studies of PEPCK with remote data collection

Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and the complex experimental infrastructure that are required remain serious obstacles to its widespread application. Here, millisecond mix-and-quench crystallography (MMQX) has been developed, which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (PEPCK) is observed with a time resolution of 40 ms. By lowering the entry barrier to time-resolved crystallography, MMQX should enable a broad expansion in structural studies of protein dynamics.

Experimental and Theoretical Studies of Dissociative Electron Attachment to Metabolites Oxaloacetic and Citric Acids

In this contribution the dissociative electron attachment to metabolites found in aerobic organisms, namely oxaloacetic and citric acids, was studied both experimentally by means of a crossed-beam setup and theoretically through density functional theory calculations. Prominent negative ion resonances from both compounds are observed peaking below 0.5 eV resulting in intense formation of fragment anions associated with a decomposition of the carboxyl groups. In addition, resonances at higher energies (3–9 eV) are observed exclusively from the decomposition of the oxaloacetic acid. These fragments are generated with considerably smaller intensities. The striking findings of our calculations indicate the different mechanism by which the near 0 eV electron is trapped by the precursor molecule to form the transitory negative ion prior to dissociation. For the oxaloacetic acid, the transitory anion arises from the capture of the electron directly into some valence states, while, for the citric acid, dipole- or multipole-bound states mediate the transition into the valence states. What is also of high importance is that both compounds while undergoing DEA reactions generate highly reactive neutral species that can lead to severe cell damage in a biological environment.

Millisecond Mix-and-Quench Crystallography (MMQX) Enables Time-Resolved Studies of PEPCK With Remote Data Collection

Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and complex experimental infrastructure required remain serious obstacles to widespread application. We have developed Millisecond Mix-and-Quench crystallography (MMQX), which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase is observed with a time resolution of 40 ms. MMQX, by lowering the entry barrier to time-resolved crystallography, should enable broad expansion in structural studies of protein dynamics.

Rethinking the Citric Acid Cycle: Connecting Pyruvate Carboxylase and Citrate Synthase to the Flow of Energy and Material

In 1937, Sir H. A Krebs first published the Citric Acid Cycle, a unidirectional cycle with carboxylic acids. The original concept of the Citric Acid Cycle from Krebs’ 1953 Nobel Prize lecture illustrates the unidirectional degradation of lactic acid to water, carbon dioxide and hydrogen. Here, we add the heart lactate dehydrogenase•proton-linked monocarboxylate transporter 1 complex, connecting the original Citric Acid Cycle to the flow of energy and material. The heart lactate dehydrogenase•proton-linked monocarboxylate transporter 1 complex catalyses the first reaction of the Citric Acid Cycle, the oxidation of lactate to pyruvate, and thus secures the provision of pyruvic acid. In addition, we modify Krebs’ original concept by feeding the cycle with oxaloacetic acid. Our concept enables the integration of anabolic processes and allows adaption of the organism to recover ATP faster.

Effect of Fungi on Metabolite Changes in Kimchi During Fermentation

The purpose of this study is to investigate the effect of fungi on kimchi metabolites during fermentation. A gas chromatography-mass spectrometry (GC-MS) based metabolite profiling approach in combination with principal component analysis (PCA) is performed to differentiate metabolites produced by fungi or bacteria. To avoid bacterial growth, kimchi is treated with 100 μg/mL of ampicillin every three days from 30 to 50 days of fermentation. The relative content of the major fungi at 50 days of fermentation, between the control group and the ampicillin treatment group, was not significantly different. The administration of ampicillin changed the metabolites in kimchi by affecting the growth of kimchi bacteria. Based on the pattern of change of each metabolite, the changed metabolites are grouped into four categories: (1) metabolites produced or consumed by fungi, (2) metabolites involving both fungi and bacteria, (3) metabolites produced or consumed by bacteria, and (4) metabolites of undetermined origin. Alanine, thymine, galacturonic acid, and malonic acid can be regarded as the metabolites produced by fungi between 30 and 50 days of fermentation. In contrast, malic acid, oxaloacetic acid, galactitol, glucose, and mannitol are presumed to be the metabolites mainly consumed by fungi. This study is meaningful as the first study conducted by inhibiting growth of bacteria to identify the metabolites contributed by fungi or bacteria in the kimchi fermentation process. These results could be used to make customized kimchi that controls the production of desired metabolites by selectively controlling the formation of microbial communities in the kimchi industry.

A blood metabolomics study of metabolic variations in Inner Mongolia white cashmere goats under shortened and natural photoperiod conditions

This study investigated metabolic variations by using gas chromatography – mass spectrometry (GC–MS)-based metabolomics in the blood of Inner Mongolia white cashmere goats under shortened and natural photoperiod conditions. Twenty-four female (non-pregnant) Inner Mongolia white cashmere goats aged 1–1.5 yr with similar live weights (mean, 20.36 ± 2.63 kg) were randomly allocated into two groups: a natural daily photoperiod group (NDPP group: 10–16 h light, n = 12) and a short daily photoperiod group (SDPP group: 7 h light:17 h dark, n = 12). In this study, we found that a SDPP promoted the blood metabolic perturbations based on the GC–MS-based metabolomics investigation, and nine metabolites were related to a SDPP. Compared with the NDPP group, the contents of serine, oxaloacetic acid, xylose, l-3,4-dihydroxyphenylalanine, and xanthosine significantly were up-regulated, whereas the contents of carnitine, 1,3-diaminopropane, indole-3-acetic acid, and l-kynurenine were significantly down-regulated in the SDPP group. The different metabolites could contribute to the regulation mechanisms of promoting cashmere growth of goats in the SDPP group.

Catalytic oxidative dehydrogenation of malic acid to oxaloacetic acid

Here we report the oxidative dehydrogenation of malic acid to oxaloacetic acid, a key precursor in the fabrication of amino acids, over Pt–Bi/C catalysts.