1H, 13C, 15N resonance assignment of the enzyme KdgF from Bacteroides eggerthii

To fully utilize carbohydrates from seaweed biomass, the degradation of the family of polysaccharides known as alginates must be understood. A step in the degradation of alginate is the conversion of 4,5-unsaturated monouronates to 4-deoxy-L-erythro-5-hexoseulose catalysed by the enzyme KdgF. In this study BeKdgF from Bacteroides eggerthii from the human gut microbiota has been produced isotopically labelled in Escherichia coli. Here the 1H, 13C, and 15N NMR chemical shift assignment for BeKdgF is reported.


Biological context
There is a growing demand for new biomass globally. A recent report by EIT Climate-KIC concluded that by 2050 the biomass usage in Europe is expected to increase 70−150%, which current biomass sources cannot sustain (Material Economics 2021). A currently underutilised biomass is seaweed. Seaweed is a fast-growing plant that does not require arable land, fresh water, or fertilizer (Enquist-Newman et al. 2014). Seaweed therefore has the potential to be a sustainable source of biomass e.g. for the production of chemical commodities, like biofuels. Alginate composes 30−60% of the polysaccharides found in seaweed. Other carbohydrates include mainly mannitol and glucan, which, unlike alginate, can readily be used in current industry (Enquist-Newman et al. 2014).
Alginates are a family of linear polysaccharides found in brown seaweed and bacteria (Gorin and Spencer 1966;Haug et al. 1967). Alginate consists of (1 → 4)-linked β-Dmannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G) arranged in sequences of M, G, or alternating MG blocks (Pawar and Edgar 2012). Alginate is degraded to 4,5-unsaturated monouronates by alginate lyases (Kim et al. 2011). The monouronates are converted to 4-deoxy-Lerythro-hexoseulose uronic acid (DEH), which is reduced to the key metabolite 2-keto-3-deoxygluconate (KDG) by DEH reductase (Preiss and Ashwell 1962). The conversion of monouronates to DEH is catalysed by the enzyme KdgF, a late step of alginate degradation (Hobbs et al. 2016). For understanding the catalytic mechanism and protein dynamics of KdgF further insight is needed.

Construct design
Genomic DNA was isolated from a culture of Bacteroides eggerthii DSM 20,697 purchased from Deutsche Samlung von Mikroorganismen und Zellkulturen (Germany) using Microbiome DNA Purification Kit (Invitrogen) according to manufacturer's specifications. The gene encoding BeKdgF (UniProt: R5JNH6) was amplified using a modified Phusion High-Fidelity DNA polymerase with primers designed for USER cloning (Salomonsen et al. 2014). BeKdgF was cloned into the pET15b-USER vector by the restriction sites Ndel and BamHI, which extended BeKdgF with an N-terminal His-tag (MGSSHHHHHHGS) resulting in 126 amino acid residues in total, and the full-length construct was used for the resonance assignment. The resulting plasmid (pET15b-USER-BeKdgF) was verified by sequencing (GATC Biotech, Germany).

Sample preparation
For resonance assignment 15 N-13 C-labelled BeKdgF from B. eggerthii was produced by recombinant expression in Escherichia coli.
The cells were harvested by centrifugation (10 min, 4 °C, 5000×g), the supernatant was discarded, and the pellet resuspended in ice cold lysis buffer (50 mM HEPES, 300 mM NaCl, pH 7.7) along with one cOmplete™ EDTAfree protease inhibitor tablet (Roche). The resuspended cells were lysed using sonication for 10 min. using a Branson Sonifier equipped with a microtip. The cell lysate was centrifuged (30 min, 4 °C, 16000×g) and the supernatant sterile filtered (0.22 μm filter).
BeKdgF was purified with affinity chromatography using Ni 2+ -resin. A column was filled with 3 mL cOmplete His-tag Purification Resin (Roche), and the resin was first rinsed with ethanol (20%, 20 mL) and ultrapure water (20 mL), before being equilibrated with 20 mL lysis buffer. The lysate was loaded onto the column and the column was washed with lysis buffer (3 × 7.5 mL) and washing buffer (50 mM HEPES, 300 mM NaCl, 20 mM imidazole, pH 7.7, 8 × 7.5 mL). The protein was eluded using elution buffer (50 mM HEPES, 300 mM NaCl, 300 mM imidazole, pH 7.7) and stored at 4 °C. The enzyme molecular size and purity were assessed using SDS-PAGE.
The buffer was exchanged to the buffer used for NMR data collection (25 mM Na 2 HPO 4 , 50 mM NaCl, pH 7.2) using a VivaSpin column (5 kDa cut-off, Sartorius) and the sample was then concentrated to a volume of ≈ 130 μL on an Amicon Ultra centrifugal filter (3 kDa cut-off). The concentrated protein sample was transferred to a 3 mm NMR tube and 99.9% D 2 O (10 v/v%) (Sigma Aldrich) was added. The BeKdgF concentration in the NMR sample was calculated to 430 μM using an extinction coefficient of 10,095 M −1 cm −1 (Gasteiger et al. 2005) and measuring absorbance at 280 nm (NanoDrop One Microvolume UV-Vis spectrophotometer, Thermo Fisher Scientific). The N-terminal His-tag did not interfere negatively the NMR work thus removal of the Histag was not attempted.

NMR experiments
All NMR spectra were recorded at 20 °C on a Bruker Avance III HD 800 MHz spectrometer using a 5 mm Z-gradient CP-TCI (H/C/N) cryogenic probe at the NV-NMR-Center/ Norwegian NMR Platform (NNP) at the Norwegian University of Science and Technology (NTNU). 1 H signals were internally referenced to the water signal, and 13 C and 15 N signals were indirectly referenced to the water signal based on absolute frequency ratios (Zhang et al. 2003).
Spectra were analysed using CARA (Computer Aided Resonance Assignment) version 1.8.4.2 (Keller 2004). A dihedral angle analysis based on the measured backbone and side-chain chemical shifts was made using TALOS-N (Shen and Bax 2013).

Assignment and data deposition
Here we report the backbone and side-chain assignment of BeKdgF from B. eggerthii. The 15 N-HSQC spectrum of BeKdgF with the assigned resonances is shown in Fig. 1. The backbone assignment is almost complete (H N , H α , C α , N, and C′ > 92%). The five unassigned residues (H52, F53, P86, D87, and V88) define short two sections in BeKdgF, which may be ascribed to multiple conformations in intermediate exchange, enhance relaxation or fast exchange regime. The side-chain assignment is partially complete (side-chain H and C ≈ 59.3%). The unassigned 1 H, 13 C, 15 N resonance assignment of the enzyme KdgF from Bacteroides eggerthii 1 3 smaller peaks in the 1 H-15 N HSQC are due to impurities of the sample. The amino acid residue R108 has an unusual chemical shift as the H N , C β , and H β chemical shifts are significantly lower than expected (Ulrich et al. 2008). The chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank (BMRB) under the accession number 51288.
Secondary structural propensity was evaluated by investigating secondary chemical shift values of BeKdgF. The chemical shift deviations of C α and C β from the random-coil values for each residue (Wishart et al. 1995) were calculated, and the results can be seen in Fig. 2. Generally, the C α chemical shifts of BeKdgF are lower than the predicted random-coil values, whereas the C β values are higher. This indicates that BeKdgF generally consists of β-sheets.
The probability of secondary structural elements was calculated based on the dihedral angle analysis using TALOS-N (Shen and Bax, 2013). The results can be seen in Fig. 3. The dihedral angle analysis shows that 50% of the amino acid residues in BeKdgF form β-strands and the rest random coils with a possibility of a three amino acid residue α-helix present. The β-sheets content is consistent with the three previously reported structures of KdgF from the organisms Yersinia enterocolitica and Halomonas sp (Hobbs et al. 2016).
The 1 H, 13 C, 15 N resonance assignment of BeKdgF has been presented. The assignment gives the possibility of further investigation of BeKdgF with NMR spectroscopy. Future functional studies will include metal ion interactions, pH titration, and protein dynamics. Understanding the biological role of KdgF can aid the industrial use of alginates extracted from seaweed as biomass.

Conflict of interest
The authors declare that they have no conflict of interest.

Ethical approval
The experiments conducted do not violate any ethical principles.
Consent for publication All authors declare that they consent to the publication of this paper.
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