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Swenson, C. V.

Publications and source records attributed to Swenson, C. V..

2 recordsLinked to original sources
bioRxiv2025

Monitoring monomer-specific acyl-tRNA levels in cells with PARTI

We describe a new assay that reports directly on the acylation state of a user-chosen tRNA in cells. We call this assay 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI). It relies on high-resolution mass spectrometry identification of the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. Here we develop the PARTI workflow and apply it to understand three recent observations related to the cellular incorporation of non--amino acid monomers into protein: (1) the origins of the apparent selectivity of translation with respect to {beta}2-hydroxy acid enantiomers; (2) the activity of PylRS variants for benzyl derivatives of malonic acid; and (3) the apparent inability of N-Me amino acids to function as ribosome substrates in living cells. Using the PARTI assay, we also provide direct evidence for the cellular production of 2,3-diacylated tRNA in certain cases. The ease and simplicity of the PARTI workflow should benefit ongoing efforts to study and improve the cellular incorporation of non--amino acid monomers into proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/633079v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f3b8a0org.highwire.dtl.DTLVardef@de400dorg.highwire.dtl.DTLVardef@13f17faorg.highwire.dtl.DTLVardef@13f2c0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗
bioRxiv2022

Regioselective control of biocatalytic C-H activation and halogenation

Biocatalytic C-H activation has the potential to merge enzymatic and synthetic strategies for bond formation. FeII/KG-dependent halogenases are particularly distinguished for their ability both to control selective C-H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how regioselectivity and chain length selectivity are achieved. We now report the crystal structure of the HalB and HalD, revealing the key role of the substrate-lid in positioning the substrate for C4 vs C5 chlorination and recognition of lysine vs ornithine. Targeted engineering of the substrate-binding lid further demonstrates that these selectivities can be altered or switched, showcasing the potential to develop halogenases for biocatalytic applications.

biochemistry↗