Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation, metabolism, and survival in vivo.

Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase's enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.

Rate of dilution and redox ratio influence the refolding efficiency of recombinant fungal dehydrogenases.

Dehydrogenases from fungi are attracting attention as industrial biocatalysts due to their high activity and chiral selectivity. However, these enzymes form insoluble aggregates when overexpressed in E. coli, limiting their industrial application. In the present study, we report the systematic development of a refolding process for selected, industrially relevant fungal dehydrogenases, viz., formate dehydrogenase from Candida boidinii (CbFDH) and formate and alcohol dehydrogenases from Geotrichum candium (GcFDH and GcADH, respectively). We first employed a screen to evaluate the effects of different variables on refolding including the buffer system, additives, and rate of dilution. The extent of refolding was determined by enzyme assays, circular dichroism, and tryptophan fluorescence. Our results showed that glycerol and reducing environment are essential for refolding of these dehydrogenases. Further, slow dilution of solubilized protein over 16 h dramatically improved the recovery of refolded enzymes compared to rapid dilution. The importance of slow dilution was further confirmed in a 10-fold scaled-up refolding trial. Overall, we demonstrate a robust method for refolding of fungal dehydrogenases, thus improving their availability for various biocatalytic applications.