bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.11.10.687318

Enabling regiospecific di-halogenation in one-pot reactions using an engineered single-component flavin-dependent tryptophan halogenase

Li, H.·Huang, J.-W.·Dai, S.·Feng, D.·Liu, J.·Zhang, N.·Guo, Y.·Chen, C.-C.·Guo, R.-T.
View original source ↗Export metadata ↓

Abstract

Installation of halogen atoms to organic compounds through flavin-dependent halogenases (FDHs) catalyzed reactions has emerged as an attractive strategy in organic biochemistry and synthetic biology. A cyanobacterial FDH termed AetF that contains FDH and flavin reductase in the same polypeptide chain could become a useful tool enzyme as the need of including a reductase is omitted. Notably, AetF exploits a unique substrate-interaction network to consecutively brominate tryptophan (Trp) on C5 and then C7 to generate di-brominated Trp. In this study, structure-based engineering is used to eliminate the C7 halogenation capacity of AetF. The resulting variant AetF-AIF that contains three residue substitutions mainly catalyzes bromination or iodination on C5 of Trp. We also show that combining AetF-AIF in tandem with the wild type AetF enables the generation of heterogeneously halogenated Trp in a one-pot reaction. The final products, 5-Br-7-I-Trp or 5-I-7-Br-Trp depending on the addition order of halides, account for a high ratio in the reaction mixture without additional purification step. These results display the potentials of single-component Trp-FDHs in a wide application of the halogenation reactions. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/687318v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@101bd34org.highwire.dtl.DTLVardef@12ca797org.highwire.dtl.DTLVardef@aee280org.highwire.dtl.DTLVardef@1984907_HPS_FORMAT_FIGEXP M_FIG A single-component flavin-dependent halogenase that catalyzes di-bromination of tryptophan was engineered as a mono-halogenase and coupled in a one-pot reaction for the synthesis of heterogeneously halogenated tryptophan. C_FIG

Source connections

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Li, H., Huang, J.-W., Dai, S., Feng, D., Liu, J., Zhang, N., Guo, Y., Chen, C.-C., Guo, R.-T.. 2025-11-11. Enabling regiospecific di-halogenation in one-pot reactions using an engineered single-component flavin-dependent tryptophan halogenase. https://doi.org/10.1101/2025.11.10.687318

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A feed-forward UHRF1 read-write mechanism supports H3 multi- mono-ubiquitination and DNA methylation maintenance at CpG-sparse regions

The epigenetic inheritance of mammalian DNA methylation requires DNMT1 and its E3 ligase cofactor UHRF1. At newly replicated chromatin, UHRF1 recognition of hemi-methylated DNA and histone H3 N-terminal tails directs catalysis of H3K14, H3K18, and/or H3K23 mono-ubiquitination to recruit DNMT1. While it is appreciated that UHRF1 can deposit multiple mono-ubiquitin marks on a single H3 tail and that DNMT1 recognizes this state through tandem ubiquitin interacting motifs, the mechanism that promotes successive ubiquitination and the biological function of multi-mono-ubiquitination are unknown. Here, we show that UHRF1 directly binds its mono-ubiquitinated H3 products through a previously uncharacterized LGDDSL loop in Tudor 2 of its tandem Tudor domain (TTD) to promote further ubiquitin deposition. Disruption of this ubiquitin reading activity impairs H3 multi-mono-ubiquitination and accelerates DNA methylation loss within late-replicating, CpG-sparse genomic regions that are characteristic of partially methylated domains (PMDs) in cancer and aging cells. These methylation defects overlap those observed by disruption of UHRF1 ubiquitin ligase activity, providing convergent evidence that both writing and reading of H3 ubiquitination support CpG-sparse DNA methylation maintenance. Together, these findings establish a feed-forward ubiquitin read-write mechanism that generates multi-mono-ubiquitinated H3 and safeguards DNMT1-dependent DNA methylation maintenance at vulnerable genomic regions of the mammalian methylome.

molecular biology↗
bioRxiv2026

Calcium dysregulation amplifies fibrotic responses to TGFβ in human Friedreich's ataxia fibroblasts

Friedreich's ataxia (FA) is an inherited disease caused by loss of frataxin (FXN) and characterized by neurodegeneration and fatal cardiomyopathy. Cardiac fibrosis contributes to cardiomyopathy by stiffening the heart wall, yet the underlying mechanisms remain unknown. Here, we investigated pro-fibrotic predisposition in FA patient-derived fibroblasts, focusing on the role of cytosolic calcium (Ca) in TGF{beta}-driven fibroblast-to-myofibroblast transition (FMT). We found pro-fibrotic transcriptional priming in FA fibroblasts, alongside elevated expression of genes controlled by the Ca-responsive transcription factor NFAT. Upon FMT, FA myofibroblasts showed amplified induction of pro-fibrotic (CCN2, NOX4) and suppression of anti-fibrotic (CCN3) genes, which were inversely correlated with residual FXN. Mechanistically, FA fibroblasts exhibited elevated cytosolic Ca and strongly downregulated expression of the Na-Ca exchanger NCX1, which directly correlated with FXN. Furthermore, NCX1 inhibition in control fibroblasts recapitulated FA Ca phenotypes, whereas NCX1 transduction in FA fibroblasts normalized Ca dynamics and blunted CCN2 induction in FMT. These findings highlight NCX1 as a modulator of fibrotic reprogramming in FA and identify Ca dyshomeostasis as an intrinsic mechanism of fibrosis that could be targeted therapeutically.

molecular biology↗
bioRxiv2026

Stromal CTHRC1 protects the valvular interstitium from macrophage-associated inflammatory remodeling and calcification

Background: Calcific aortic valve disease (CAVD) is characterized by progressive inflammatory and fibrocalcific remodeling. Although valvular interstitial cells (VICs) are generally considered to drive fibrosis and osteogenic remodeling, whether injury-activated VICs mount endogenous protective responses that preserve the valvular interstitial microenvironment and restrain calcification remains unknown. Methods: We performed spatial transcriptomic profiling of aortic valves in a mouse model of endothelial injury-induced CAVD to define early injury-responsive programs within the valvular interstitium. The cellular origin and spatial distribution of candidate protective factors were examined by immunohistochemistry and lineage tracing, and their relevance to human disease was assessed using stenotic aortic valves. The functional role of CTHRC1 was investigated using genetic Cthrc1 deficiency combined with longitudinal hemodynamic assessment, histological analysis, and spatial transcriptomic profiling. Results: Spatial transcriptomics identified Cthrc1 as a prominent component of an early injury-induced stromal response in the expanding valvular interstitium. CTHRC1 was strongly expressed in activated VICs within thickened murine valve leaflets and human stenotic aortic valves. Lineage tracing demonstrated that the expanded VIC population arose predominantly from PDGFR{beta}+ resident interstitial cells, with minimal endothelial contribution. Despite comparable early hemodynamic responses to endothelial injury, Cthrc1 deficiency exacerbated chronic valvular calcification. Spatial profiling of Cthrc1-deficient valves revealed pronounced interstitial accumulation of galectin-3+ foamy macrophages, accompanied by mitochondrial respiratory-chain signature loss and cell death-associated pathway activation. These findings indicate that transient CTHRC1 induction after endothelial injury defines an endogenous stromal protective response that preserves the valvular interstitial microenvironment and limits macrophage-associated tissue injury and subsequent dystrophic calcification. Conclusions: Injury-activated VICs are not merely effectors of pathological remodeling, but can engage an endogenous tissue-protective response through CTHRC1. These findings identify a previously unrecognized stromal defense mechanism linking endothelial injury to macrophage-associated inflammatory remodeling and dystrophic calcification and suggest CTHRC1-dependent stromal protection as a potential therapeutic axis for limiting CAVD progression.

molecular biology↗