Biology subjects

Weber, K. R.

Publications and source records attributed to Weber, K. R..

3 recordsLinked to original sources

bioRxiv2026

Mechanistic insights into redox activity and catalytic determinants of the haloarchaeal flavin-dependent oxidoreductase HvFdR

Members of the FAD-dependent oxidoreductase family (IPR050260) play diverse and key roles in maintaining cellular redox balance, yet the functions of many distinct subgroups within this family remain unknown. Here, we define the biochemical and physiological functions of the Haloferax volcanii flavin-dependent oxidoreductase HvFdR (HVO_2345; fdr), a haloarchaeal member of a previously uncharacterized IPR050260 subgroup. HvFdR binds FAD and catalyzes NAD(P)H oxidase, diaphorase and ferredoxin reductase activities, with a kinetic preference for NADPH over NADH and catalytic properties that are strongly influenced by oxygen availability. Under stoichiometric conditions, HvFdR mediates reverse electron transfer to NADP, suggesting that intracellular nicotinamide nucleotide pools regulate electron flow bidirectionally. Consistent with this reversibility, HvFdR bound-FAD exhibits a low midpoint redox potential (-413 mV), supporting its capacity to function as an electron donor. Deletion of fdr impairs growth and elevates intracellular NADPH levels, consistent with a role for HvFdR in maintaining NADP(H) homeostasis. Conserved residues K47 and Y323 are identified as determinants of HvFdR electron transfer activity and may function as a regulatory gate that modulates electron flow while limiting excessive H2O2 production under aerobic conditions. Together, these findings establish HvFdR as an oxygen-responsive flavin-dependent oxidoreductase that contributes to cellular redox homeostasis and provides functional insight into a previously uncharacterized subgroup of the IPR050260 family.

biochemistry↗

bioRxiv2026

Lysine acetylation-mediated regulation of ferredoxin and ferredoxin reductase redox-active proteins in Haloferax volcanii

Lysine acetylation is an evolutionarily conserved, post-translational modification that regulates metabolism and protein function, yet its role in archaeal electron transfer systems remains poorly understood. Here, we investigated lysine acetylation of the 2Fe-2S ferredoxin HvFdx (HVO_2995) and its flavin-dependent oxidoreductase HvFdR (HVO_2345) partner in the halophilic archaeon Haloferax volcanii. Genetic and biochemical analyses established HvFdx as an essential 2Fe-2S ferredoxin with a midpoint redox potential of -385 mV. Lysine acetylation of HvFdx was found to occur primarily at K119, a residue positioned near the [Fe-S] cluster interface, and to modulate electron transfer capacity without impacting Fe-S cluster incorporation, midpoint potential, or protein abundance. In contrast, HvFdR was found lysine acetylated at multiple sites in a manner consistent with a non-enzymatic mechanism that resulted in altered flavin binding, enzymatic activity, and thermal stability. Lysine acetylation of HvFdx was found to stimulate electron flow from HvFdR as measured by an anaerobic NADPH [->] HvFdR [->] HvFdx [->] DCIP assay. 3D structural modeling, proteomic, biochemical, and genetic assays suggest the haloarchaeal GNAT-family acetyltransferase homolog HVO_2874 as a candidate enzyme associated with HvFdx lysine acetylation and optimal growth of H. volcanii. Together, these findings demonstrate that lysine acetylation differentially regulates archaeal redox-active proteins and functions as an important mechanism coordinating redox metabolism in H. volcanii.

microbiology↗

bioRxiv2025

Revisiting synthetic lethality of Gcn5-related N-acetyltransferase (GNAT) family mutations in Haloferax volcanii

Lysine acetylation is a post-translational modification that occurs in all domains of life, highlighting its evolutionary significance. Previous genome comparison identified three Gcn5-related N-acetyltransferase (GNAT) family members as lysine acetyltransferase homologs (Pat1, Pat2, and Elp3) and two deacetylase homologs (Sir2 and HdaI) in the halophilic archaeon Haloferax volcanii, with elp3 and pat2 proposed as a synthetic lethal gene pair. Here we advance these findings by performing single and double mutagenesis of elp3 with the pat1 and pat2 lysine acetyltransferase gene homologs. Genome sequencing and PCR screens of these strains reveal successful generation of{Delta} elp3, {Delta}pat1{Delta}elp3, and{Delta} pat2{Delta}elp3 mutant strains. Although these mutant strains exhibited a reduced growth rate compared to the parent, they remained viable. Overall, this study provides genetic evidence that elp3 and pat2, while impacting cell growth, are not a synthetic lethal gene pair as previously reported. IMPORTANCEHere we reveal by whole genome sequencing that the GNAT family gene homologs elp3 and pat2 can be deleted in the same H. volcanii strain. Beyond the targeted deletions, minimal differences between the parent and{Delta} elp3 {Delta}pat2 mutant were observed suggesting that suppressor mutations are not responsible for our ability to generate this double mutant strain. Elp3 and Pat2, thus, may not share as close a functional relationship as implied by earlier study. Our finding is significant as Elp3 is thought to function in acetylation in tRNA modification, while Pat2 likely functions in the lysine acetylation of proteins.

genetics↗