Livia Regina Manzine

Formation of a Ternary Complex for Selenocysteine Biosynthesis in Bacteria

Background: Selenoprotein biosynthesis requires the interaction of tRNASec and specific enzymes that drive the synthesis of selenocysteine. Results: Formation of a molecular complex of selenophosphate synthetase, selenocysteine synthase, and tRNASec was identified and characterized. Conclusion: The ternary complex formation is necessary for selenoprotein synthesis. Significance: Our findings demonstrate the formation of a ternary complex and provide a possible scenario for selenium metabolism in bacteria. The synthesis of selenocysteine-containing proteins (selenoproteins) involves the interaction of selenocysteine synthase (SelA), tRNA (tRNASec), selenophosphate synthetase (SelD, SPS), a specific elongation factor (SelB), and a specific mRNA sequence known as selenocysteine insertion sequence (SECIS). Because selenium compounds are highly toxic in the cellular environment, the association of selenium with proteins throughout its metabolism is essential for cell survival. In this study, we demonstrate the interaction of SPS with the SelA-tRNASec complex, resulting in a 1.3-MDa ternary complex of 27.0 ± 0.5 nm in diameter and 4.02 ± 0.05 nm in height. To assemble the ternary complex, SPS undergoes a conformational change. We demonstrated that the glycine-rich N-terminal region of SPS is crucial for the SelA-tRNASec-SPS interaction and selenoprotein biosynthesis, as revealed by functional complementation experiments. Taken together, our results provide new insights into selenoprotein biosynthesis, demonstrating for the first time the formation of the functional ternary SelA-tRNASec-SPS complex. We propose that this complex is necessary for proper selenocysteine synthesis and may be involved in avoiding the cellular toxicity of selenium compounds.

Assembly stoichiometry of bacterial selenocysteine synthase and SelC (tRNAsec)

In bacteria selenocysteyl–tRNAsec (SelC) is synthesized by selenocysteine synthase (SelA). Here we show by fluorescence anisotropy binding assays and electron microscopical symmetry analysis that the SelA–tRNAsec binding stoichiometry is of one tRNAsec molecule per SelA monomer (1:1) rather than the 1:2 value proposed previously. Negative stain transmission electron microscopy revealed a D5 pointgroup symmetry for the SelA–tRNAsec assembly both with and without tRNAsec bound. Furthermore, SelA can associate forming a supramolecular complex of stacked decamer rings, which does not occur in the presence of tRNAsec. We discuss the structure–function relationships of these assemblies and their regulatory role in bacterial selenocysteyl–tRNAsec synthesis.

Biophysical and biochemical studies of a major endoglucanase secreted by Xanthomonas campestris pv. campestris.

Endoglucanases are the main cellulolytic enzymes secreted by the bacterium Xanthomonas campestris pv. campestris (Xcc). The major endoglucanase exported by this bacterium into an external milieu is an enzyme XccCel5A, which belongs to GH5 family subfamily 1 and is encoded by the gene engXCA. We purified XccCel5A using ammonium sulfate precipitation followed by size exclusion chromatography and identified it by zymogram analysis. Circular dichroism and fluorescence spectroscopy studies showed that XccCel5A is stable in a wide pH range and up to about 55°C and denatures at the higher temperatures. The optimal conditions for enzyme activity were identified as T=45°C and pH=7.0. Under the optimum conditions the catalytic efficiency (kcat/KM) of the enzyme was determined as 5.16×10(4)s(-1)M(-1) using carboxymethylcellulose (CMC) as a substrate. Our SAXS studies revealed extended tadpole-shape molecular assembly, typical for cellulases, and allowed to determine an overall shape of the enzyme and a relative position of the catalytic and cellulose binding domains.

Structural insights into β-glucosidase transglycosylation based on biochemical, structural and computational analysis of two GH1 enzymes from Trichoderma harzianum

β-glucosidases are glycoside hydrolases able to cleave small and soluble substrates, thus producing monosaccharides. These enzymes are distributed among families GH1, GH2, GH3, GH5, GH9, GH30 and GH116, with GH1 and GH3 being the most relevant families with characterized enzymes to date. A recent transcriptomic analysis of the fungus Trichoderma harzianum, known for its increased β-glucosidase activity as compared to Trichoderma reesei, revealed two enzymes from family GH1 with high expression levels. Here we report the cloning, recombinant expression, purification and crystallization of these enzymes, ThBgl1 and ThBgl2. A close inspection of the enzymatic activity of these enzymes surprisingly revealed a marked difference between them despite the sequence similarity (53%). ThBgl1 has an increased tendency to catalyze transglycosylation reaction while ThBgl2 acts more as a hydrolyzing enzyme. Detailed comparison of their crystal structures and the analysis of the molecular dynamics simulations reveal the presence of an asparagine residue N186 in ThBgl2, which is replaced by the phenylalanine F180 in ThBgl1. This single amino acid substitution seems to be sufficient to create a polar environment that culminates with an increased availability of water molecules in ThBgl2 as compared to ThBgl1, thus conferring stronger hydrolyzing character to the former enzyme.

An improved purification procedure for Leishmania RNA virus (LRV).

Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations.

Biochemical characterization, low-resolution SAXS structure and an enzymatic cleavage pattern of Bl Cel48 from Bacillus licheniformis

Economic sustainability of modern biochemical technologies for plant cell wall transformations in renewable fuels, green chemicals, and sustainable materials is considerably impacted by the elevated cost of enzymes. Therefore, there is a significant drive toward discovery and characterization of novel carbohydrate-active enzymes. Here, the BlCel48 cellulase from Bacillus licheniformis, a glycoside hydrolase family 48 member (GH48), was functionally and biochemically characterized. The enzyme is catalytically stable in a broad range of temperatures and pH conditions with its enzymatic activity at pH5.0 and 60°C. BlCel48 exhibits high hydrolytic activity against phosphoric acid swollen cellulose (PASC) and bacterial cellulose (BC) and significantly lower activity against carboxymethylcellulose (CMC). BlCel48 releases predominantly cellobiose, and also small amounts of cellotriose and cellotetraose as products from PASC hydrolysis. Small-angle X-ray scattering (SAXS) data analysis revealed a globular molecular shape and monomeric state of the enzyme in solution. Its molecular mass estimated based on SAXS data is ~77.2kDa. BlCel48 has an (αα)6-helix barrel-fold, characteristic of GH48 members. Comparative analyses of homologous sequences and structures reveal the existence of two distinct loops in BlCel48 that were not present in other structurally characterized GH48 enzymes which could have importance for the enzyme activity and specificity.

Investigation of Escherichia coli Selenocysteine Synthase (SelA) Complex Formation Using Cryo-Electron Microscopy (Cryo-EM)

Incorporation of selenocysteine (Sec U) into proteins is directed by a in-frame UGA codon in all domains of life. In Bacteria, Sec biosynthesis and incorporation involves the interaction of Selenocysteine Synthase (SelA), tRNA (SelC or tRNA Sec ), Selenophosphate Synthetase (SPS), a specific elongation factor known as SelB and the specific mRNA structure SElenocysteine Insertion Sequence (SECIS), forming a complex molecular machinery. SelA is a homodecamer complex responsible for Ser-Sec conversion from selenophosphate delivered by SPS and seryl-tRNA sec , which differs from seryl-tRNA ser by its long variable arm and the UGA-codon. The specific mRNA sequence known as SElenoCysteine Insertion Sequence forms a hairpin-like secondary structure and is recognized by SelB for Sec incorporation in the nascent peptide [1-3]. Since selenium compounds are highly toxic in cellular environment, selenium association with proteins complexes throughout its metabolism is suggested to be essential for cell survival. However, macromolecular interactions between the different proteins have not yet been characterized.