The enzyme protochlorophyllide oxidoreductase (POR) catalyses a lightdependent step in chlorophyll biosynthesis that is essential to photosynthesis and ultimately all life on Earth. 1-3 POR, which is one of three known light-dependent enzymes, 4,5 catalyzes reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, a structural basis for POR photocatalysis has remained elusive. Here, we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex have identified multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways. As the light-driven step in the chlorophyll biosynthetic pathway (Fig. 1), the POR reaction acts as the trigger for the germination of seedlings =in plants and provokes a marked change in the morphological development of the plant. 2,3 Given this crucial biological role, POR has been the focus of numerous mechanistic and biophysical investigations. A combination of time-resolved (at the femtosecond-to-second scale) and cryogenic spectroscopy methods have provided some understanding of the mechanism of POR photocatalysis in a range of photosynthetic organisms, including cyanobacteria and plants. Picosecond excited-state dynamics in the protochlorophyllide (Pchlide) molecule are thought to result in excited state interactions between the substrate and active-site residues that are necessary to trigger the subsequent reaction chemistry. 6-12 This involves sequential transfer of a hydride equivalent from NADPH and a proton transfer from either an active site residue or solvent. Proton transfer is reliant on solvent dynamics and an implied network of extended protein motions that occur on the microsecond timescale. 13-17 Hydride transfer from NADPH is not concerted, but occurs in a stepwise manner that involves

Cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase (cy-FBP/ SBPase) plays a vital role in gluconeogenesis and in the photosynthetic carbon reduction pathway, and is thus a potential enzymatic target for inhibition of harmful cyanobacterial blooms. Here, we describe the crystal structure of cy-FBP/SBPase in complex with AMP and fructose-1,6-bisphosphate (FBP). The allosteric inhibitor AMP and the substrate FBP exhibit an unusual binding mode when in complex with cy-FBP/SBPase. Binding mode analysis suggested that AMP bound to the allosteric sites near the interface across the up/down subunit pairs C1C4 and C2C3 in the center of the tetramer, while FBP binds opposite to the interface between the horizontal subunit pairs C1C2 or C3C4. We identified a series of residues important for FBP and AMP binding, and suggest formation of a disulfide linkage between Cys75 and Cys99. Further analysis indicates that cy-FBP/ SBPase may be regulated through ligand binding and alteration of the structure of the enzyme complex. The interactions between ligands and cy-FBP/ SBPase are different from those of ligand-bound structures of other FBPase family members, and thus provide new insight into the molecular mechanisms of structure and catalysis of cy-FBP/SBPase. Our studies provide insight into the evolution of this enzyme family, and may help in the design of inhibitors aimed at preventing toxic cyanobacterial blooms. DatabaseStructural data have ben submitted to the Protein Data Bank under accession numbers 3ROJ and 3RPL.Structured digital abstract cy-FBP/SBPase and cy-FBP/SBPase bind by x-ray crystallography (View interaction).

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of a rice plants 9,311 (Oryza sativa L.) cDNA in rice plants zhonghua11 (Oryza sativa L.). The genetic engineering enabled the plants to accumulate SBPase in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO(2) assimilation of transgenic plants was significantly more tolerant to high temperature than that of wild-type plants. The analyses of chlorophyll fluorescence and the content and activation of SBPase indicated that the enhancement of photosynthesis to high temperature was not related to the function of photosystem II but to the content and activation of SBPase. Western blotting analyses showed that high temperature stress led to the association of SBPase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than that in transgenic plants. The results in this study suggested that under high temperature stress, SBPase maintained the activation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fraction and thus enhanced the tolerance of CO(2) assimilation to high temperature stress. The results suggested that overexpression of SBPase might be an effective method for enhancing high temperature tolerance of plants.

Kava (Piper methysticum Foster, Piperaceae) organic solvent‐extract has been used to treat mild to moderate anxiety, insomnia, and muscle fatigue in Western countries, leading to its emergence as one of the 10 best‐selling herbal preparations. However, several reports of severe hepatotoxicity in kava consumers led the U.S. Food and Drug Administration and authorities in Europe to restrict sales of kava‐containing products. Herein we demonstrate that flavokawain B (FKB), a chalcone from kava root, is a potent hepatocellular toxin, inducing cell death in HepG2 (LD50=15.3±0.2 µM) and L‐02 (LD50=32 µM) cells. Hepatocellular toxicity of FKB is mediated by induction of oxidative stress, depletion of reduced glutathione (GSH), inhibition of IKK activity leading to NF‐κB transcriptional blockade, and constitutive TNF‐α‐independent activation of mitogen‐activated protein kinase (MAPK) signaling pathways, namely, ERK, p38, and JNK We further demonstrate by noninvasive bioluminescence imaging that oral consumption of FKB leads to inhibition of hepatic NF‐κB transcriptional activity in vivo and severe liver damage. Surprisingly, replenishment with exogenous GSH normalizes both TNF‐α‐dependent NF‐κB as well as MAPK signaling and rescues hepatocytes from FKB‐in‐duced death. Our data identify FKB as a potent GSH‐sensitive hepatotoxin, levels of which should be specifically monitored and controlled in kava‐containing herb products.—Zhou, P., Gross, S., Liu, J.‐H., Yu, B.‐Y., Feng, L.‐L., Nolta, J., Sharma, V., Piwnica‐Worms, D., Qiu, S. X. Flavokawain B, the hepatotoxic constituent from kava root, induces GSH‐sensitive oxidative stress through modulation of IKK/NF‐κB and MAPK signaling pathways. FASEB J. 24, 4722–4732 (2010). http://www.fasebj.org

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC3.1.3.37) was increased in the transgenic rice cultivar zhonghua11 (Oryza sativa L. ssp. japonica) by overexpressing OsSbp cDNA from the rice cultivar 9311 (Oryza sativa ssp. indica). This genetic engineering enabled the transgenic plants to accumulate SBPase in chloroplasts and resulted in enhanced tolerance of transgenic rice plants to salt stress at the young seedlings stage. Moreover, CO2 assimilation in transgenic rice plants was significantly more tolerant to salt stress than in wild-type plants. The analysis of chlorophyll fluorescence and the activity of SBPase indicated that the enhancement of photosynthesis in salt stress was not related to the function of PSII but to the activity of SBPase. Western-blot analysis showed that salt stress led to the association of SBPase with the thylakoid membranes from the stroma fractions. However, this association was much more prominent in wild-type plants than in transgenic plants. Results suggested that under salt stress, SBPase maintained the activation of ribulose-1,5-bisphosphate carboxylase-oxygenase by providing more regeneration of the acceptor molecule ribulose-1,5-bisphosphate in the soluble stroma and by preventing the sequestration of Rubisco activase to the thylakoid membrane from the soluble stroma, and, thus, enhanced the tolerance of photosynthesis to salt stress. Results suggested that overexpression of SBPase was an effective method for enhanncing salt tolerance in rice.

Monoclonal antibodies targeting PD-1/PD-L1 signaling pathway have achieved unprecedented success in cancer treatment over the last few years. Atezolizumab is the first PD-L1 monoclonal antibody approved by US FDA for cancer therapy; however the molecular basis of atezolizumab in blocking PD-1/PD-L1 interaction is not fully understood. Here we have solved the crystal structure of PD-L1/atezolizumab complex at 2.9 angstrom resolution. The structure shows that atezolizumab binds the front beta-sheet of PD-L1 through three CDR loops from the heavy chain and one CDR loop from the light chain. The binding involves extensive hydrogen-bonding and hydrophobic interactions. Notably there are multiple aromatic residues from the CDR loops forming Pi-Pi stacking or cation-Pi interactions within the center of the binding interface and the buried surface area is more than 2000 Å2, which is the largest amongst all the known PD-L1/antibody structures. Mutagenesis study revealed that two hot-spot residues (E58, R113) of PD-L1 contribute significantly to the binding of atezolizumab. The structure also shows that atezolizumab binds PD-L1 with a distinct heavy and light chain orientation and it blocks PD-1/PD-L1 interaction through competing with PD-1 for the same PD-L1 surface area. Taken together, the complex structure of PD-L1/atezolizumab solved here revealed the molecular mechanism of atezolizumab in immunotherapy and provides basis for future monoclonal antibody optimization and rational design of small chemical compounds targeting PD-L1 surface.

Harmful Microcystis blooms (HMBs) seriously threaten the ecology of environments and human health. Microcystins (MCs) produced by Microcystis are powerful mediators of HMB induction and maintenance. In this study, microcystinase A (MlrA), an enzyme with MC-degrading ability, was successfully obtained at over 90% purity for the first time through overexpression in Escherichia coli K12 TB1. The obtained MlrA exhibited high stability at high temperature and under alkaline conditions, while also exhibiting a long half-life. MlrA selectively inhibited MC-producing Microcystis cultures, but had no effect on MC-nonproducing Synechocystis cultures. The inhibition mechanism of MlrA against Microcystis was investigated by evaluating the morphological and physiological characteristics of cultures. MlrA effectively degraded extracellular MCs and decreased the synthesis of intracellular MCs by causing downregulation of genes involved in the microcystin biosynthesis pathway. Concomitantly, MlrA inhibited Microcystis photosynthesis by causing the downregulated expression of important photosynthesis pathway genes and interrupting electron transport chain activities and pigment synthesis. Thus, MlrA achieved the inhibition of Microcystis growth by reducing its photosynthetic capacity and intracellular MC contents, while also degrading extracellular MCs. On the basis of these results, we propose a new paradigm to achieve the simultaneous removal of MCs and HMBs using the single enzyme characterized here.

Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170 kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.