Biology subjects

Neun, S.

Publications and source records attributed to Neun, S..

3 recordsLinked to original sources

bioRxiv2023

The biosynthesis, degradation, and function of cell wall β-xylosylated xyloglucan mirrors that of arabinoxyloglucan

O_LIXyloglucan is an abundant polysaccharide in many primary cell walls and in the human diet. Decoration of its -xylosyl side chains with further sugars is critical for plant growth, even though the sugars themselves vary considerably between species. Plants in the Ericales order--prevalent in human diets--exhibit {beta}1,2-linked xylosyl decorations. The biosynthetic enzymes responsible for adding these xylosyl decorations, as well as the hydrolases that remove them in the human gut, are unidentified. C_LIO_LIGT47 xyloglucan glycosyltransferase candidates were expressed in Arabidopsis and endo-xyloglucanase products from transgenic wall material were analysed by electrophoresis, mass spectrometry, and NMR. The activities of gut bacterial hydrolases BoGH43A and BoGH43B on synthetic glycosides and xyloglucan oligosaccharides were measured by colorimetry and electrophoresis. C_LIO_LICcXBT1 is a xyloglucan {beta}-xylosyltransferase from coffee that can modify Arabidopsis xyloglucan and restore the growth of galactosyltransferase mutants. Related VmXST1 is a weakly active xyloglucan -arabinofuranosyltransferase from cranberry. BoGH43A hydrolyses both -arabinofuranosylated and {beta}-xylosylated oligosaccharides. C_LIO_LICcXBT1s presence in coffee and BoGH43As promiscuity suggest that {beta}-xylosylated xyloglucan is not only more widespread than thought, but might also nourish beneficial gut bacteria. The evolutionary instability of transferase specificity and lack of hydrolase specificity hint that, to enzymes, xylosides and arabinofuranosides are closely resemblant. C_LI

biochemistry↗

bioRxiv2022

High throughput steady-state enzyme kinetics measured in a parallel droplet generation and absorbance detection platform

Microfluidic water-in-oil emulsion droplets are becoming a mainstay of experimental biology, where they replace the classical test tube. In most applications (e.g. in ultrahigh throughput directed evolution) the droplet content is identical for all compartmentalized assay reactions. When emulsion droplets are used for kinetics or other functional assays, though, concentration dependencies (e.g. of initial rates for Michaelis-Menten plots) are required. Droplet-on-demand systems satisfy this need but extracting large amounts of data is challenging. Here we introduce a multiplexed droplet absorbance detector which, coupled to semi-automated droplet generation, forms a tubing-based droplet-on-demand system able to generate and extract quantitative datasets from defined concentration gradients across multiple series of droplets for multiple time points. The emergence of product is detected by reading the absorbance of the droplet sets at multiple, adjustable time points (reversing the flow direction after each detection, so that the droplets pass a line scan camera multiple times). Detection multiplexing allows absorbance values at twelve distinct positions to be measured and enzyme kinetics are recorded for label-free concentration gradients (composed of about 60 droplets each, covering as many concentrations). With a throughput of around 8640 data points per hour, a 10-fold improvement compared to the previously reported single point detection method is achieved. In a single experiment, twelve full datasets of high-resolution and high accuracy Michaelis-Menten kinetics were determined to demonstrate the potential for enzyme characterization for glycosidase substrates covering a range in enzymatic hydrolysis of seven orders of magnitude in kcat/KM. The straightforward set-up, high throughput, excellent data quality, wide dynamic range that allows coverage of diverse activities suggest that this system may serve as a miniaturized spectrophotometer to for detailed analysis of study clones emerging from large-scale combinatorial experiments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/500969v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1e240b3org.highwire.dtl.DTLVardef@105ba2corg.highwire.dtl.DTLVardef@101cd63org.highwire.dtl.DTLVardef@1534a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood

The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for {beta}-glucuronidase activity. We identified SN243, a genuine {beta}-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase (GH) 3 family member. This GH family had no recorded evidence of {beta}-glucuronidases at the outset of this study, showing that a functional metagenomic approach can shed light on assignments that are currently unpredictable by bioinformatics. Kinetic analyses of SN243 characterised it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous GH3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.

biochemistry↗