Sequence specific 1H, 13C and 15N backbone resonance assignments of UVI31+ from Chlamydomonas reinhardtii

Abstract

The cDNA of UVI31+ was cloned from C. reinhardtii and expressed in E. coli from where the protein was purified to homogeneity. The purified protein exhibited beta-lactamase activity (Manuscript in preparation). However, UVI31+ has no homology with the known β-lactamases. In order to understand the structural basis of the ability of UVI31+ to hydrolyze β-lactam antibiotics, we in parallel, set out to structurally characterize it by NMR. Its β-lactamase activity in relation to the solution structure by NMR is likely to provoke deeper understanding of its mechanism and facilitate the rationalization of other functions of the protein, if any. In this endeavor, we report almost complete sequence-specific backbone (1)H, (13)C and (15)N NMR assignments of UVI31+.

A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

“…The protease activity of PI-PLC on the substrate protein UVI31+ [31], [32] was inhibited by the protease inhibitor leupeptin, while other inhibitors like AEBSF were unstable during a long incubation (Fig. 2A).…”

“…Each reaction mixture (30 µL total volume) contained 13 µM purified UVI31+ protein [31], [32] (14 kDa) and 0.2 units PI-PLC in 50 mM ammonium bicarbonate, and was incubated overnight at 37°C. The protein was then denatured by the addition of 7 µL SDS-denaturing solution (200 mM Tris-HCl pH 6.8, 8% SDS (w/v), 40% glycerol (v/v), 4% 2-mercaptoethanol (w/v), 50 mM EDTA pH 8.0, and 0.08% bromophenol blue (w/v) and heating at 100°C for 3 min.…”

UVI31+ Is a DNA Endonuclease That Dynamically Localizes to Chloroplast Pyrenoids in C. reinhardtii

“…In spite of low sequence homology (identity: 27% and similarity: 54%), C. reinhardtii UVI31+ protein shows substantial structural homology with the known tertiary structure of E. Coli BolA ([14] and unpublished observations). With this in mind and gain an insight into the BolA domain of UVI31+, we tested whether UVI31+ also causes round morphology in bacterial cells.…”

Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

“…An intriguing question is whether this ordering would also apply to the kca t values of these proteins. Previous work in our laboratory has established that the protein UVI31+ has weak β-lactamase activity, although sequence alignment with known β-lactamases has failed to identify the active site [92]. CLASP based predictions and site directed mutagenesis are underway to ascertain the active site.…”

“…Each reaction mixture (30 µl total volume) contained 13 µM of purified UVI31+ protein [92] (14 kDa) and 3 units of AP in 50 mM ammonium bicarbonate, followed by overnight incubation at 37°C. The protein was then denatured by the addition of 7 µl of SDS-denaturing solution (200 mM Tris-HCl pH 6.8, 8% SDS, 40% Glycerol, 4% 2-mercaptoethanol, 50 mM EDTA pH 8.0, and 0.08% bromophenol blue) and heating it at 100°C for 3 min.…”