Silencing of OsCV (chloroplast vesiculation) maintained photorespiration and N assimilation in rice plants grown under elevated CO2

High CO₂ concentrations stimulate net photosynthesis by increasing CO₂ substrate availability for Rubisco, simultaneously suppressing photorespiration. Previously, we reported that silencing the chloroplast vesiculation (cv) gene in rice increased source fitness, through the maintenance of chloroplast stability and the expression of photorespiration-associated genes. Because high atmospheric CO₂ conditions diminished photorespiration, we tested whether CV silencing might be a viable strategy to improve the effects of high CO₂ on grain yield and N assimilation in rice. Under elevated CO₂, OsCV expression was induced, and OsCV was targeted to peroxisomes where it facilitated the removal of OsPEX11-1 from the peroxisome and delivered it to the vacuole for degradation. This process correlated well with the reduction in the number of peroxisomes, the decreased catalase activity and the increased H₂O₂ content in wild-type plants under elevated CO₂. At elevated CO₂, CV-silenced rice plants maintained peroxisome proliferation and photorespiration and displayed higher N assimilation than wild-type plants. This was supported by higher activity of enzymes involved in NO₃⁻ and NH₄⁺ assimilation and higher total and seed protein contents. Co-immunoprecipitation of OsCV-interacting proteins suggested that, similar to its role in chloroplast protein turnover, OsCV acted as a scaffold, binding peroxisomal proteins.     

Structural Basis for Catalysis of a Tetrameric Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one-third of the world's population in its latent form. The emergence of multidrug-resistant and extensive drug-resistant strains has highlighted the need for new pharmacological targets within M. tuberculosis. The class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since it is upregulated in latent TB. Since the structure of MtFBA has not been determined and there is little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering the pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and fructose 1,6-bisphosphate at 1.5, 2.1, and 1.3 Å, respectively. Through these structures, it was discovered that MtFBA belongs to a novel tetrameric class of type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for better predictability of the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Substrates and products were observed in geometries poised for catalysis; in addition, unexpectedly, the hydroxyl-enolate intermediate of dihydroxyacetone phosphate was also captured and resolved structurally. These concise new details offer a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes.     

One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis

Rv0363c (fba), encoding Class II fructose-bisphosphate aldolase (FBA), is one of the potential drug targets identified in our laboratory based on minimal gene set concept. The wild-type enzyme overproduction in E. coli had been reported. However, the purification procedure was relatively tedious and the yield was low. In this study, five histidine codons were introduced into the 3′ end of the amplified fba fragments. The expressed C-terminal histidine-tagged Class II FBA was produced in E. coli BL21 (DE3) and easily purified using immobilized metal affinity chromatography. The purified his-tagged FBA was characterized. Its biochemical properties were compared to the non-his-tagged enzyme purified according to the previous report. Both FBAs have similar characteristics such as native/subunit molecular mass, kinetic parameters, and temperature/pH optima and stability. The C-terminal his-tagged FBA can be a surrogate for the native enzyme and used for screening of inhibitors of FBA. This developed expression system will pave the way for high-throughput screening and crystallization studies. Moreover, in this study, a colorimetric FBA assay has been simplified to facilitate the mass screening of inhibitor of FBA.     

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.