Biology subjects

Boswinkle, K.

Publications and source records attributed to Boswinkle, K..

3 recordsLinked to original sources

bioRxiv2026

The Escherichia coli Radical SAM Enzyme YhcC Substitutes for the FAD-Dependent Oxidase Activity of MnmC in 5-Methylaminomethyl-2-Thiouridine tRNA Modification Under Anaerobic Conditions

tRNA wobble uridines are heavily modified to influence anticodon-codon pairing and tune anticodon stem-loop structure for efficient, accurate translation. Many bacteria and some archaea modify wobble uridines with either a 5-carboxymethylaminomethyl (cmnm5) or 5-methylaminomethyl (mnm5) group, often together with a 2-thio (s2) moiety. Bacteria utilize the conserved MnmEG complex to produce cmnm5U, which is further converted to mnm5U by non-orthologous enzymes in different lineages. Escherichia coli uses the bifunctional enzyme MnmC to demodify cmnm5U to nm5U and subsequently methylate nm5U to mnm5U whereas Bacillus subtilis relies on the radical SAM (rSAM) enzyme MnmL and the stand-alone methylase MnmM. Although E. coli and related bacteria encode homologs of MnmL, the function of the E. coli homolog, YhcC, remained unknown. Here, we show that YhcC is required for cmnm5s2 U demodification in vivo during anaerobic growth, whereas the equivalent MnmC-dependent reaction requires O2 and occurs only aerobically. In vitro, purified [4Fe-4S]-reconstituted YhcC binds tRNA and catalyzes nm5s2U-tRNA synthesis from cmnm5s2U-tRNA. Together, these results define the previously unknown function of the E. coli rSAM enzyme YhcC and demonstrate that it replaces MnmC under anaerobic conditions to generate nm5s2U. These parallel pathways reveal how E. coli maintains synthesis of a critical wobble-base modification under both aerobic and anaerobic growth conditions.

biochemistry↗

bioRxiv2026

Moraxella catarrhalis HemW is a Heme-binding Radical SAM Enzyme

Moraxella catarrhalis is an emerging human respiratory pathogen responsible for a significant proportion of childhood otitis media and exacerbations of chronic obstructive pulmonary disease. Recent transposon sequencing analysis identified yggW (renamed as hemW), an uncharacterized gene, as essential for M. catarrhalis growth under iron-limiting conditions, mimicking host-imposed nutritional immunity. HemW is annotated as a putative radical S-adenosylmethionine (SAM) enzyme and belongs to the HemN-like subfamily, but its biochemical properties remain unclear. Here, we report on the first experimental characterizations of McHemW. Our bioinformatic analysis confirmed its evolutionary relationship with the putative heme-binding radical SAM enzyme EcHemW in Escherichia coli. Using biochemical and spectroscopic approaches, we demonstrate that McHemW contains a catalytically active [4Fe-4S] cluster and binds heme in vitro. These findings support a functional role for McHemW as a putative heme chaperone, highlighting it as a potential target for disrupting iron metabolism in this clinically important pathogen.

biochemistry↗

bioRxiv2023

Control of Biofilm Formation by an Agrobacterium tumefaciens Pterin-Binding Periplasmic Protein Conserved Among Pathogenic Bacteria

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are formed via a non-essential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering cdGMP breakdown and dampening its synthesis. Pterins are excreted and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon that is conserved amongst multiple Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a new pterin-responsive regulatory mechanism that controls biofilm formation and related cdGMP-dependent phenotypes in A. tumefaciens and is found in multiple additional bacterial pathogens. SIGNIFICANCEBiofilms are bacterial communities attached to surfaces, physiologically distinct from free-living cells, and a common cause of persistent infections. Here we define the mechanism of a novel biofilm regulatory system based on excreted metabolites called pterins, that is conserved within a wide range of Gram-negative bacteria, including multiple pathogens of animals and plants. The molecular mechanism of pterin-dependent regulation is reported including structural determination of several members of a new family of pterin-binding proteins. Pterins are produced across all domains of life and mechanistic insights into this regulatory circuit could lead to new advances in antibiofilm treatments.

microbiology↗