Journal: Frontiers in Plant Science

Article Title: Engineering and Modulating Functional Cyanobacterial CO 2 -Fixing Organelles

doi: 10.3389/fpls.2018.00739

Figure Lengend Snippet: Synthetic operon design and heterologous expression of synthetic β-carboxysomes in Escherichia coli . (A) Schematic representation of the molecular organization of the natural β-carboxysome operons spread in five loci within the Syn7942 genome and the synthetic β-carboxysome operons (pLFbC901 and pLFbC601) inserted in the E. coli expression vector pETM11. Locus tags are indicated. (B) Thin-section TEM images of E. coli WT and cells expressing β-carboxysome proteins using pLFbC601 and pLFbC901 plasmids. Arrows indicate β-carboxysome-like structures with polyhedral shapes observed in pLFbC601 and pLFbC901 E. coli cells. (C) In vivo carbon fixation assays of E. coli WT, pLFbC901, and pLFbC601 cells, indicating the CO -fixing activity of synthetic β-carboxysomes. A relatively higher CO -fixing activity was determined in E. coli pLFbC901 cells compared with that of E. coli pLFbC601 cells. (D) SDS-PAGE of the total cell extracts of the recombinant E. coli pLFbC901 and the E. coli WT cells. Putative β-carboxysome proteins (RbcL, RbcS, CcmM, CcmK2, CcmK4) were identified based on their molecular weights. The presence of these β-carboxysome protein blocks was further confirmed by proteomic analysis ( Supplementary Table ). The gel image was spliced based on Supplementary Figure .

Article Snippet: The operons were inserted into either the pETM11 vector digested at the EcoRI and XhoI sites or the pAM2991 vector [a gift from Susan Golden, Addgene plasmid # 40248 ( )] at the EcoRI and BamHI sites.

Techniques: Expressing, Plasmid Preparation, In Vivo, Activity Assay, SDS Page, Recombinant

Journal: Scientific Reports

Article Title: A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase

doi: 10.1038/s41598-018-31259-y

Figure Lengend Snippet: ( a ) Overall structure of the La IHA dimer found in the asymmetric unit, the protomers are colored green and cyan and shown in two different orientations rotated by 90 degrees. Manganese and highlighted residues a location of active sites in the dimer. ( b ) Isatinate and benzyl benzoate in the substrate binding pocket of La IHA. Isatinate is coordinating bidentate directly to the manganese. Parts of the pocket, residues Trp61 and Trp59, are contributed by the opposing monomer via the hairpin swap. Key binding pocket residues of the La IHB binding pocket (grey) are functionally conserved. ( c ) Catalytically important residue in the active site of La IHA: benzyl benzoate. The corresponding site of La IHA: benzyl benzoate is superposed (transparent). The manganese is found in octahedral coordination similarly to that described in . Gln219 resides in a double conformation and only partially coordinates to the manganese. Also, W1025 is found in a double conformation (denoted A and B in Fig. 1). Note that Asp75 is coordinating bidentate in La IHA:isatinate while monodentate in La IHA: benzyl benzoate. ( d ) The isatin hydrolysis by La IHA (circles) and Rs IHA (triangles) follows Michaelis-Menten kinetics, and the parameters are collected in f). ( e ) Both La IHA and Rs IHA display strong manganese dependency. ( f ) Kinetic parameters of La IHA and Rs IHA collected with previous values from La IHB and activated mutant La IHB S225C. All measurements in ( d ) and ( e ) were performed in triplets.

Article Snippet: The Rs IHA (UniProtKB: Q8XYC3) was ordered from Genscript and inserted into the expression vector pET-M11.

Techniques: Binding Assay, Residue, Mutagenesis