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bioRxiv · 10.64898/2026.05.13.723581

Biophysical and enzymatic comparison of Bacillus safensis and Bacillus subtilis malate dehydrogenase (MDH) enzymes

Zafiropoulo, H. R.·Thomas, J. E.·Cortez, N. R.·Apostol, K.·de Sa, A.·Khosravi, R.·Moore, L.·Berndsen, C. E.·Bibel, B.
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Abstract

Species of Bacillus bacteria including Bacillus safensis and Bacillus subtilis are finding increasing uses in biotechnology and bioremediation, thanks in part to their metabolic robustness. Malate dehydrogenase (MDH) is at the heart of central metabolism and thus a better understanding of Bacillus MDH proteins could aid in the optimization of these applications. MDH of Bacillus spp. belong to the lactate dehydrogenase (LDH)-like class of MDHs, otherwise known as the MDH3 class. Despite wide prevalence in nature among prokaryotes and archaea, this typically homotetrameric class is understudied compared to the MDH1 and MDH2 classes found in eukaryotes. We therefore recombinantly expressed and purified MDH proteins from two societally relevant Bacillus spp.-B. safensis and B. subtilis-and characterized them biophysically (via Size Exclusion Chromatography-Small Angle X-ray Scattering (SEC-SAXS) and Differential Scanning Fluorimetry (DSF)) and enzymatically (via spectroscopic activity assays). As expected based on their high sequence identity, the two MDH orthologs had similar properties in most regards, including a tetrameric structure and high susceptibility to substrate inhibition. However, we uncovered differences in conditional thermal stability, in addition to subtle differences in enzymatic activity that offer insight into the workings of LDH-like MDH. Summary statementMalate dehydrogenase (MDH) is a fundamental metabolic enzyme, from microbes to mammals, yet comparably little is known about microbial MDH, especially MDH of the tetrameric MDH3 class. We compare the biophysical and enzymatic properties of two such enzymes from the societally relevant bacterial species Bacillus subtilis and Bacillus safensis, offering useful insight with potential biotechnological implications.

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Zafiropoulo, H. R., Thomas, J. E., Cortez, N. R., Apostol, K., de Sa, A., Khosravi, R., Moore, L., Berndsen, C. E., Bibel, B.. 2026-05-14. Biophysical and enzymatic comparison of Bacillus safensis and Bacillus subtilis malate dehydrogenase (MDH) enzymes. https://doi.org/10.64898/2026.05.13.723581

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