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Acta Cryst. (2014). A70, C1820
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Coat proteins of several isometric viruses consist of two domains, a disordered N-terminal R-domain consisting of several positively charged residues and a shell (S) domain characterized by a jelly roll β-barrel structure. The three-dimensional structure of Sesbania mosaic virus (SeMV), a T=3 plant virus, has been determined at 3 Å resolution. The full length coat protein, when expressed in E. coli, assembles into T=3 icosahedral shells (VLPs) resembling native virus particles. In the present investigations, the role of N-terminal R domain in the assembly of VLPs was explored by replacing the R domain with a presumably globular domain (SeMV-P10) and other intrinsically disordered (SeMV-P8, and SeMV-VPg) SeMV encoded domains. The R domain was also replaced with the non-viral globular B-domain of Staphyloccocus aureus protein A. These domains were of nearly the same size as that of the R-domain. Most of the chimeric coat proteins, when expressed in E.coli, formed VLPs, which could be purified by ultra-centrifugation. The purified VLPs were examined by transmission electron microscopy (TEM), which suggested that a fraction of the expressed proteins could assemble into T=3 VLPs, although often, the particles were heterogeneous. Interestingly, the SeMV NΔ65B CP could also be purified by Ni-NTA chromatography as a dimer which assembled into T=1 VLPs under crystallization conditions. The structure of NΔ65B-CP VLPs revealed that the assembled particles were devoid of divalent metal ions at the canonical site and there was no density corresponding to the B domain. However, the S domain could be superimposed on that of SeMV NΔ65VLPs determined earlier. The other VLPs- SeMVNΔ65P10 CP, SeMVNΔ65P8 CP and SeMVNΔ65VPg could not be crystallized because of their heterogeneity. These studies suggest a subtle interplay between the length, sequence and structure of the R-domain polypeptide and the assembly of particles.

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Acta Cryst. (2014). A70, C1821
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As a part of exploration of novel bacterial genes coding for insecticidal proteins, we have initiated structural and functional studies on oxalate decarboxylase from Photorhabdus luminescens, a symbiotic bacteria inhabiting guts of nematodes that parasitize insects. Oxalate decarboxylases (OXDC) are enzymes that catalyze the conversion of oxalate to formate and carbondioxide (CO2) in the presence of manganese and dioxygen. Structures of OXDC in complex with ethylene glycol (EDO), EDO/formate (FMT) and FMT/CO2 were determined at resolutions of 1.97Å, 2.36Å and 2.5Å, respectively. The asymmetric unit of these crystals contained a trimeric molecule. A protomer of the enzyme consists of two β-barrel domains belonging to the cupin family of proteins. All the three ligand bound structures of OXDC resemble the closed form of OXDC from B. subtilis reported earlier. Comparison of these structures with the open and closed forms of B. subtilis OXDC indicated that the conformation of Glu166 (corresponding to Glu162 of B. subtilis) in cupin domain I but not in domain II is in a conformation suitable to function as the catalytic base/acid and hence only domain I may be catalytically active. It is observed that the hydrogen bonding interaction between Arg95 and Thr169 of cupin domain I is essential for the positioning of Glu166 for catalysis. A corresponding threonine residue is absent in cupin domain II. An analysis of the similarities and differences between OXDC structures from P. luminescens and other reported bacterial OXDC structures and oxalate oxidase from Hordeum vulgare has been carried out with the view of understanding substrate and functional specificities of these enzymes. The structure provides the molecular framework required to investigate the mode of action of the enzyme, which may be a suitable candidate for developing P. luminescens as a bio-insecticide against plant pests.

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Acta Cryst. (2014). A70, C1822
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Diaminopropionate ammonia lyase (DAPAL) is a non-stereo specific fold-type II pyridoxal 5' phosphate (PLP) dependent enzyme that catalyzes the conversion of both D/L isoforms of the nonstandard amino acid Diaminopropionate (DAP) to pyruvate and ammonia. DAP is important for the synthesis of nonribosomal peptide antibiotics such as viomycin and capreomycin. Earlier structural studies on EcDAPAL bound to a reaction intermediate (aminoacrylate) suggested that the enzyme follows a two base mechanism, where Asp120 and Lys77 function as general bases to abstract proton from D-DAP and L-DAP respectively. A novel disulfide was observed near the active site, although its functional significance was not clear. In the present study, structural and biochemical characterization of active site mutants Asp120 (Asp120Asn/Ser/Thr/Cys) and Lys77 (Lys77His/ Thr/Ala/Val) of EcDAPAL has been carried out. Reduction of catalytic efficiency (Kcat/Km) of D120N EcDAPAL for D-DAP by 140 fold and presence of the uncatalyzed ligand at the active site in the crystal structure suggested that Asp120 indeed abstracts proton from D-DAP. Lys77, which was speculated to be important for proton abstraction from L DAP, however seemed to be crucial for PLP binding only. Presence of non-covalently bound PLP in the L77W mutant and occurence of both the ketoenamine, enolimine forms of internal aldimine in L77R mutant provided an in depth insight into the unique chemistry of internal aldimine formation in PLP dependent enzymes. To investigate the role of the novel disulfide bond near the active site, C265 and C291 were mutated to Serine. Studies on these mutants show that this disulfide bond gives additional stability to the protein and might regulate the entry of substrates to the active site. Thus, these studies provide deeper insights into the reaction mechanism of EcDAPAL, representing the overall reaction mechanism followed by several other fold-type II PLP pendent enzymes.
Keywords: PLP; DAPAL; DAP.
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