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This project aims to solve the structure of the crenarchaeal Sulfolobus acidocaldarius enzyme malate synthase. Other known malate synthase enzymes have been found to require a magnesium ion in the active site to carry out catalytic activities, but a study reported that S. acidocaldarius malate synthase does not require magnesium. This suggests a novel mechanism for this enzyme. Additionally, the mature S. acidocaldarius protein is approximately 100 residues larger than any other structurally characterized malate synthase. It has also been reported to form a dimer, while previously solved structures have only displayed monomeric, trimeric, and hexameric arrangements. We plan to determine the structure experimentally. However, major advances in the accuracy of protein structure prediction were made recently by AlphaFold, an artificial intelligence system developed by DeepMind, which has revolutionized the field and has largely solved the protein folding problem. A similar AI system, RoseTTAFold, developed by David Baker's lab at the University of Washington, has been made publicly available. Here, we report our analysis of the structure of this protein, predicted using both of these algorithms and of a predicted structural model for the dimeric form of the enzyme using ClusPro. Our results strongly support a conserved catalytic mechanism requiring magnesium, which is common with all previously solved malate synthase isoforms. [ABSTRACT FROM AUTHOR]

A thorough search for the development of innovative drugs to treat tuberculosis, especially considering the urgent need to address developing drug resistance, we report here a synthetic series of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o) as potent anti-tubercular agents. These morpholino-indolizines were synthesized by reacting 4-morpholino pyridinium salts, with various electron-deficient acetylenes to afford the ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o). All synthesized intermediate and final compounds are characterized by spectroscopic methods such as 1 H NMR, 13 C NMR and HRMS and further examined for their anti-tubercular activity against the M. tuberculosis H37Rv strain (ATCC 27294-American type cell culture). All the compounds screened for anti-tubercular activity in the range of 6.25-50 μM against the H37Rv strain of Mycobacterium tuberculosis. Compound 5g showed prominent activity with MIC 99 2.55 μg/mL whereas compounds 5d and 5j showed activity with MIC 99 18.91 μg/mL and 25.07 μg/mL, respectively. In silico analysis of these compounds revealed drug-likeness. Additionally, the molecular target identification for Malate synthase (PDB 5CBB) is attained by computational approach. The compound 5g with a MIC 99 value of 2.55 μg/mL against M. tuberculosis H37Rv emerged as the most promising anti-TB drug and in silico investigations suggest Malate synthase (5CBB) might be the compound's possible target., (© 2024 John Wiley & Sons Ltd.)

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the β-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in β galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)