Biology subjects

Rosenzweig, A. C.

Publications and source records attributed to Rosenzweig, A. C..

3 recordsLinked to original sources

bioRxiv2026

Thiooxazole Formation on a Nontypeable Haemophilus influenzae Virulence Factor Requires a Mixed-Valent Diiron Cofactor

The multinuclear nonheme iron-dependent oxidative enzyme (MNIO) family employs a multi-iron cofactor to catalyze a range of post-translational modifications (PTMs) in the biosynthesis of ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. While significant progress has been made toward understanding the range of chemical transformations performed by MNIOs, the nature of the iron cofactor has only been investigated in one instance. Here, we examine the MNIO involved in oxazolin biosynthesis to gain further insight into the metallocofactors employed by this impressive family of enzymes. Oxazolin, a RiPP virulence factor from nontypeable Haemophilus influenzae, contains six copper-binding 5-thiooxazole groups installed by the MNIO HvfB. Weak interactions between HvfB and its required partner protein, HvfC, motivated genetic fusion of the two proteins, which yielded an effective mimic of the protein complex with high enzymatic activity. While HvfB binds up to three iron ions, concerted EPR, ENDOR, and Mossbauer spectroscopic characterization of the active protein reveals that accumulation of a mixed-valent diiron(II/III) cluster correlates with 5-thiooxazole product formation. This oxidation state is attained only in the presence of HvfC, revealing a new role for the partner protein in modulating the iron cofactor. Site-directed mutagenesis of metal-coordinating residues was used to probe the function of the third iron-binding site. This work clarifies the nature of the active iron cofactor for oxazolin maturation, providing a second example of a mixed-valent diiron oxidase in RiPP biosynthesis. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/739211v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@8cabdorg.highwire.dtl.DTLVardef@1364ba5org.highwire.dtl.DTLVardef@1a059f4org.highwire.dtl.DTLVardef@71c9fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Differentiating 5-thiooxazoles from oxazolone-coupled thioamides in RiPP natural products

Conversion of cysteine residues to 5-thiooxazole moieties by multinuclear nonheme iron-dependent oxidative enzymes (MNIOs) is a prevalent modification in ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. However, this post-translational modification (PTM) is difficult to distinguish from MNIO-produced oxazolone-coupled thioamides, such as those present in the RiPP methanobactin. The RiPP virulence factor oxazolin contains six copper-binding heterocycles installed by an MNIO. Here, we reassign these PTMs, originally described as oxazolones/thioamides, as 5-thiooxazoles on the basis of detailed comparative chemical and structural characterization of oxazolin and methanobactin. These data establish a benchmark for differentiating these two PTMs in newly discovered RiPPs. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/730506v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1fc7a4eorg.highwire.dtl.DTLVardef@c1b8baorg.highwire.dtl.DTLVardef@46cf36org.highwire.dtl.DTLVardef@ce1fea_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Prokaryotic mechanosensitive channels mediate copper influx

Copper is an essential micronutrient in all kingdoms of life, requiring a meticulous balance between acquisition and toxic overload. While copper import in eukaryotes has been investigated extensively, few prokaryotic copper importers have been identified, leading to the notion that cytoplasmic copper uptake is unnecessary in prokaryotes. Here we report that mechanosensitive channels are key players in prokaryotic copper import. Deletion of the gene encoding the E. coli small mechanosensitive channel, EcMscS, leads to significantly reduced copper influx. Conversely, overexpression of EcMscS leads to increased copper influx, elevated intracellular copper content, and renders cells hypersensitive to copper. Furthermore, specific channel blockers and competing permeating ions inhibit EcMscS copper conductance, lowering intracellular copper accumulation and alleviating copper hypersensitivity. These findings extend beyond E. coli, as other prokaryotic small mechanosensitive channels of bacterial and archaeal origin also facilitate copper influx. Taken together, these results uncover a previously unknown moonlighting function for mechanosensitive channels as a pathway for prokaryotic copper uptake.

microbiology↗