Marine Goux

Sucrose phosphorylases are essential enzymes regulating sucrose metabolism, and it has been shown that a loop rearrangement is essential to their catalytic cycle. Crystal structures of only six sucrose phosphorylase enzymes are available. Here, we present the crystal structure of a sucrose phosphorylase from a proteobacterium, Alteromonas mediterranea , at 2.15 Å resolution. The available sucrose phosphorylase structures have shown that an important conformational change occurs during the catalytic cycle or upon mutagenesis. Interestingly, our data present clear indications of the two major conformations in the same crystal.

Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer regioselectively glucose from sucrose, the donor substrate, onto acceptors like flavonoids to form glycoconjugates and hence modulate their solubility and bioactivity. Here, we report for the first time the structure of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea ( Am SP) and its enzymatic properties. Kinetics of sucrose hydrolysis and transglucosylation capacities on (+)-catechin were investigated. Wild-type enzyme (AmSP-WT) displayed high hydrolytic activity on sucrose and was devoid of transglucosylation activity on (+)-catechin. Two variants, Am SP-Q353F and Am SP-P140D catalysed the regiospecific transglucosylation of (+)-catechin: 89% of a novel compound (+)-catechin-4′-O-α-D-glucopyranoside (CAT-4’) for AmSP-P140D and 92% of (+)-catechin-3′-O-α-D-glucopyranoside (CAT-3’) for AmSP-Q353F. The compound CAT-4’ was fully characterized by NMR and mass spectrometry. An explanation for this difference in regiospecificity was provided at atomic level by molecular docking simulations: Am SP-P140D was found to preferentially bind (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4’ while the binding mode in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3’.

A new Ba SP variant Q345F/P134D controls (+)-catechin glucosylation regioselectivity by modifying stabilisation interactions in the catalytic pocket of the enzyme.

Epidermal growth-factor receptor (EGFR) is involved in cell growth and proliferation and is over-expressed in malignant tissues. Although anti-EGFR-based immunotherapy became a standard of care for patients with EGFR-positive tumors, this strategy of addressing cancer tumors by targeting EGFR with monoclonal antibodies is less-developed for patient diagnostic and monitoring. Indeed, antibodies exhibit a slow blood clearance, which is detrimental for positron emission tomography (PET) imaging. New molecular probes are proposed to overcome such limitations for patient monitoring, making use of low-molecular-weight protein scaffolds as alternatives to antibodies, such as Nanofitins with better pharmacokinetic profiles. Anti-EGFR Nanofitin B10 was reformatted by genetic engineering to exhibit a unique cysteine moiety at its C-terminus, which allows the development of a fast and site-specific radiolabeling procedure with ¹⁸F–4-fluorobenzamido-N-ethylamino-maleimide (¹⁸F–FBEM). The in vivo tumor targeting and imaging profile of the anti-EGFR Cys–B10 Nanofitin was investigated in a double-tumor xenograft model by static small-animal PET at 2 h after tail-vein injection of the radiolabeled Nanofitin ¹⁸F–FBEM–Cys–B10. The image showed that the EGFR-positive tumor (A431) is clearly delineated in comparison to the EGFR-negative tumor (H520) with a significant tumor-to-background contrast. ¹⁸F–FBEM–Cys–B10 demonstrated a significantly higher retention in A431 tumors than in H520 tumors at 2.5 h post-injection with a A431-to-H520 uptake ratio of 2.53 ± 0.18 and a tumor-to-blood ratio of 4.55 ± 0.63. This study provides the first report of Nanofitin scaffold used as a targeted PET radiotracer for in vivo imaging of EGFR-positive tumor, with the anti-EGFR B10 Nanofitin used as proof-of-concept. The fast generation of specific Nanofitins via a fully in vitro selection process, together with the excellent imaging features of the Nanofitin scaffold, could facilitate the development of valuable PET-based companion diagnostics.

Objectives To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. Results The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). Conclusion The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.