Biology subjects

Leimkuehler, S.

Publications and source records attributed to Leimkuehler, S..

2 recordsLinked to original sources

bioRxiv2025

Protein dynamics affect O2-stability of Group B -hydrogenase from Thermosediminibacter oceani

In the pursuit of sustainable green energy generation, [FeFe]-hydrogenases have attracted significant attention due to their ability to catalyze hydrogen production. However, the sensitivity of these enzymes to O2 is a major obstacle for their application as biocatalysts in energy conversion technologies. In the search for an O2-stable [FeFe]-hydrogenase, we identified the hydrogenase ToHydA from Thermosediminibacter oceani that belongs to the rarely characterized Group B (M2a) [FeFe]-hydrogenases. Our findings demonstrate that ToHydA exhibits remarkable O2-stability, even under prolonged O2 exposure. By characterizing site-directed mutagenesis variants, we found that the highly conserved proton-transporting cysteine protects H-cluster from O2-induced degradation by forming Hinact state. The additional cysteine residue in the TSCCCP motif of ToHydA, a feature unique to Group B (M2a) [FeFe]-hydrogenases, enhances the flexibility of that motif and facilitates the formation of the Hinact state. Moreover, ToHydA possesses unique features, including the formation of an unusual Hinact resting state that distinguishes the enzyme from other [FeFe]-hydrogenases. Our atomistic molecular dynamics simulations reveal a previously unrecognized cluster of hydrophobic residues centered around the proton-transporting cysteine-bearing loop. This structural feature appears to be a common molecular characteristic in hydrogenases that form the O2-protected Hinact state. By exploiting these molecular features of ToHydA, future research can aim to rationally design hydrogenases that combine high catalytic activity with enhanced O2 stability, to develop more efficient and durable catalysts. Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/643706v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@96b4aorg.highwire.dtl.DTLVardef@62f367org.highwire.dtl.DTLVardef@59f170org.highwire.dtl.DTLVardef@fa2fd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Genome-wide CRISPR screens reveal APR-246 (Eprenetapopt) triggers ferroptosis and inhibits iron-sulfur cluster biogenesis

The mechanisms by which cells respond and adapt to oxidative stress are largely unknown but are key to developing a rationale for cancer therapies that target antioxidant pathways. APR-246 is a mutant-p53 targeted therapeutic currently under clinical investigation in myeloid dysplastic syndrome (MDS) and acute myeloid leukemia1. Whilst the mechanism of action of APR-246 is thought to be reactivation of wild-type p53 activity through covalent modification of cysteine residues in the core domain of mutant-p53 protein2,3, here we report that the anti-neoplastic capacity of APR-246 lies predominantly in the conjugation of free cysteine. Genome-wide CRISPR perturbation screening, metabolite profiling and proteomics in response to APR-246 treatment in mutant-p53 cancer cells highlighted the role of GSH and mitochondrial metabolism in determining APR-246 efficacy. APR-246 sensitivity was increased through loss of key enzymes in mitochondrial one-carbon metabolism, SHMT2 and MTHFD1L, due to diminished glycine supply for de novo GSH synthesis. Critically, we show that APR-246 induces iron-dependent, apoptotic machinery-independent cell death, ferroptosis. Whole-cell proteomics analyses indicated an upregulation of proteins involved in iron-sulfur cluster biogenesis (eg. FDX1). GSH, acetyl-CoA and NADH levels were also depleted in APR-246 treated cells. Importantly, we found that APR-246 inhibits iron-sulfur cluster biogenesis in the mitochondria of cancer cells through cysteine conjugation. This work not only details novel determinants of APR-246 activity in cancer cells, but also provides a clinical roadmap for targeting antioxidant pathways in tumours - beyond targeting mutant-p53 tumours. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/398867v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@11155f8org.highwire.dtl.DTLVardef@1231cb7org.highwire.dtl.DTLVardef@324881org.highwire.dtl.DTLVardef@136146f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗