Biology subjects

Enemark-Rasmussen, K.

Publications and source records attributed to Enemark-Rasmussen, K..

3 recordsLinked to original sources

bioRxiv2026

TFA-defluorinating microbial consortium achieved with analogue enrichment

Trifluoroacetate (TFA) is an ultra-short chain perfluoroalkyl substance (PFAS) and a pollutant of global significance; it is persistent and ubiquitous in the environment across compartments, including food, drinking water, animals, and humans. Concentrations in the environment are rising, concerns over the toxicity of the compound are increasing, and there are currently no efficient ways to remediate TFA pollution, as conventional methods are ineffective at scale under environmental conditions. Microorganisms possessing dehalogenating enzymes targeting the structurally analogous compound monofluoroacetate (MFA) present a potential solution to degrade TFA microbiologically. Due to previously demonstrated dehalogenation of difluoroacetate (DFA) by dehalogenases, the promiscuity of such enzymes was hypothesized to be extendable to TFA as well. In this study cultures obtained from an environmental sample of PFAS contaminated wastewater sludge were incubated in M9 minimal media supplied with 33 mM acetate and either 33 mM TFA, DFA, or MFA in an iterative manner, followed by testing in a 30-day experiment. Free fluoride (F-) was measured at 5 timepoints using a fluoride ion specific electrode (ISE). One culture designated MFA1C significantly degraded MFA, DFA, and TFA (two-way ANOVA mixed model, p < 0.05). MFA1C released 11.2 {+/-} 0.7 M F- which constitutes approximately 0.01% of the potential fluoride release from the total 33 mM TFA applied and with the greatest defluorination activity in the first 9 days of incubation. Fluoride release from TFA and formation of DFA and MFA was confirmed with 19F-NMR. Genetic sequencing and protein-ligand modelling identified two candidate dehalogenase enzymes from Achromobacter denitrificans and Stenotrophomonas sp., respectively, with potential capacity to degrade TFA. The findings of this study are a crucial first step towards future potential bioremediation of TFA, and are, to the best of our knowledge, the first to identify a TFA-degrading consortium of microorganisms.

microbiology↗

bioRxiv2023

High-yield enzymatic synthesis of mono- and trifluorinated alanine enantiomers

Fluorinated amino acids are a promising entry point for incorporating new-to-Nature chemistries in biological systems. Hence, novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers. To this end, the alanine dehydrogenase from Vibrio proteolyticus and the diaminopimelate dehydrogenase from Symbiobacterium thermophilum were applied to the in vitro production of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, an alanine racemase from Streptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in the {beta}-elimination of fluorine from the monosubstituted fluoroalanine. The in vitro enzymatic cascade based on the dehydrogenases of V. proteolyticus and S. thermophilum included a cofactor recycling system, whereby a formate dehydrogenase from Pseudomonas sp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. Moreover, the selected dehydrogenases were also able to catalyze the conversion of trifluoropyruvate into trifluorinated alanine, as a first-case example of biocatalysis with amino acids carrying a trifluoromethyl group.

synthetic biology↗

bioRxiv2023

Identification of non-conventional small molecule degraders and stabilizers of squalene synthase

Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/543387v1_figu1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1645e05org.highwire.dtl.DTLVardef@58ad70org.highwire.dtl.DTLVardef@1e2c57aorg.highwire.dtl.DTLVardef@112fcbd_HPS_FORMAT_FIGEXP M_FIG C_FIG Squalene synthase (SQS) is an essential enzyme in the mevalonate pathway whose abundance and activity control cholesterol biosynthesis and homeostasis. Although catalytic inhibitors of SQS have been developed to attenuate cholesterol, none so far have been approved for therapeutic use. Herein we sought to develop SQS degraders using targeted protein degradation (TPD) as an approach to lower overall cellular cholesterol content. We found that KY02111, a small molecule ligand of SQS, could selectively cause SQS to degrade in a proteasome-dependent manner. In contrast, compounds based on the same scaffold linked to E3 ligase recruiting ligands led to SQS stabilization. Whole cell proteomic analysis found KY02111 to reduce only the levels of SQS, while lipidomic analysis determined that KY02111 treatment concomitantly reduced cellular cholesteryl ester content. SQS stabilizers were shown to shield SQS from its natural turnover without recruiting their matching E3 ligase. Our work shows that degradation of SQS is possible despite a challenging biological setting and lays the groundwork for future development of either SQS degrading or stabilizing probes.

cell biology↗