Functional metagenomic screening identifies an unexpected β-glucuronidase

Abstract

The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a millionmembered metagenomic library at ultrahigh throughput in microfluidic droplets for βglucuronidase activity. We identified SN243, a genuine β-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase (GH) 3 family member. This GH family contains only one recently added β-glucuronidase, 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 >10 7 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.

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

“…The values determined for k cat (0.4 s -1 ) and K M (21.3 mM) differ only minimally from the kinetic data obtained in microtiter plate (k cat = 0.4 s -1 , K M = 23.0 mM). 22 Comparison of the datasets from all twelve detection points (Figures S5-S12, Supporting Information), which were obtained from three different sets of droplets (each feeding four detection points) proved reliable reproducibility of the data. Mean values and their standard deviation averaged from the twelve datasets for the hydrolysis of p NP-β-Xyl resulted in k cat = 0.4 ± 0.04 s -1 and K M = 21.4 ± 2.2 mM with the highest relative difference of 10% in k cat and 15% in K M .…”

“…SN243 was expressed and purified as described in Neun et al 22 Kinetics were determined in 100 mM Tris pH 8.0, 150 mM NaCl at room temperature. For kinetic measurements in droplets, the following concentrations were used: injection of 500 μM p NP-β-D-glucuronide ( p NP-β-GlcA) into 5 nM SN243; 100 mM p NP-β-D-galacturonide ( p NP-β-GalA) into 25 nM SN243; 400 mM p NP-β-D-glucopyranoside ( p NP-β-Glc) or p NP-β-D-xylopyranoside ( p NP-β-Xyl) into 1 μM SN243; 400 mM p NP-β-D-galactopyranoside ( p NP-β-Gal) or p NP-α-L-arabinofuranoside ( p NP-α-Ara f ) into 10 μM SN243.…”

“…Enzymatic reactions. SN243 was expressed and purified as described in Neun et al 22 Kinetics were determined in 100 mM Tris pH 8.0, 150 mM NaCl at room temperature. For kinetic measurements in droplets, the following concentrations were used: injection of 500 μM…”

“…Progress in the development of microfluidic tools [12][13][14][15][16][17][18] for the functional screening of enzyme libraries at ultrahigh throughput now allows us to routinely screen millions of enzyme variants and identify active variants in directed evolution and functional metagenomic projects. [19][20][21][22] By contrast, the methods used to profile newly discovered enzymes with kinetic studies remain mostly unchanged, requiring a large number of tests for full and quantitative enzyme characterization, including steady-state or pre-steady-state kinetic assays, thermostability assessments, substrate specificity and enzymatic activity under different experimental conditions (e.g. in the presence of additives and/or inhibitors).…”

“…For Michaelis-Menten kinetics obtained from plate measurements, parameters are indicated for the fit of one dataset per reaction as well as standard errors with a 95% confidence interval. Kinetic data from plate reader measurements were taken from Neun et al22…”