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Now showing items 1 - 7 of 7

  • High speed X-ray analysis of plant enzymes at room temperature

    Xia, Liqun   Rajendran, Chitra   Ruppert, Martin   Panjikar, Santosh   Wang, Meitian   Stoeckigt, Joachim  

    X-ray measurements at room temperature (295 K) deliver high quality data sets with unprecedented speed (<2 min), as shown for crystallized raucaffricine-O-β-d-glucosidase (RG), its mutant RG-Glu186Gln and several ligand complexes of the enzyme which participates in alkaloid biosynthesis in the plant Rauvolfia. The data obtained are compared with data sets measured under typical cryo conditions (100 K). Under both conditions, density maps are highly comparable and favor the described protocol for room temperature measurements, potentially paving the way for future crystallographic studies capturing biosynthetic pathway intermediates.
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  • Structures of Alkaloid Biosynthetic Glucosidases Decode Substrate Specificity

    Xia, Liqun   Ruppert, Martin   Wang, Meitian   Panjikar, Santosh   Lin, Haili   Rajendran, Chitra   Barleben, Leif   St?ckigt, Joachim  

    Two similar enzymes with different biosynthetic function in one species have evolved to catalyze two distinct reactions. X-ray structures of both enzymes help reveal their most important differences. The Rauvolfia alkaloid biosynthetic network harbors two O-glucosidases: raucaffricine glucosidase (RG), which hydrolyses raucaffricine to an intermediate downstream in the ajmaline pathway, and strictosidine glucosidase (SG), which operates upstream. RG converts strictosidine, the substrate of SG, but SG does not accept raucaffricine. Now elucidation of crystal structures of RG, inactive RG-E186Q mutant, and its complexes with ligands dihydro-raucaffricine and secologanin reveals that it is the "wider gate" of RG that allows strictosidine to enter the catalytic site, whereas the "slot-like" entrance of SG prohibits access by raucaffricine. Trp392 in RG and Trp388 in SG control the gate shape and acceptance of substrates. Ser390 directs the conformation of Trp392. 3D structures, supported by site-directed mutations and kinetic data of RG and SG, provide a structural and catalytic explanation of substrate specificity and deeper insights into O-glucosidase chemistry.
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  • Air cooling analysis and design of impacted flexible actuator’s servo controller based on PLECS

    Zhu, Mingjun   Xia, Liqun   Hu, Yixue   Zhang, Wei   Ji, Duohong   Song, Xin   Abdul Amir, H.F.   Khiew, P.S.  

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  • DNA Glycosylase Activity Assay Based on Streptavidin Paramagnetic Bead Substrate Capture

    Xia, Liqun   O'Connor, Timothy R.  

    A method to detect DNA glycosylase activity is described. The substrate used was an oligodeoxyribonucleotide with a unique hypoxanthine base, but has general application to any DNA glycosylase or endonuclease. The oligodeoxyribonucleotide was labeled at the 5′ end with 32P and at the 3′ end with a biotin linkage and annealed to a complementary oligodeoxyribonucleotide. The hypoxanthine base was excised in solution using the MPG protein, a human DNA glycosylase. Following cleavage of the phosphodiester linkage by NaOH, the oligodeoxyribonucleotide was attached to streptavidin-coated, paramagnetic beads. Binding of the labeled oligodeoxyribonucleotide to the beads was indicative of the kinetics of the reaction. As a control, half of the reaction products were loaded on to a denaturing polyacrylamide gel. Comparable values for steady-state kinetic constants were obtained using both methods. This nonelectrophoretic technique is a rapid assay of DNA glycosylase activity for both purified proteins and crude extracts. This method can be directly adapted for high-throughput techniques.
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  • Human 3-Methyladenine-DNA Glycosylase: Effect of Sequence Context on Excision, Association with PCNA, and Stimulation by AP Endonuclease

    Xia, Liqun   Zheng, Li   Lee, Hyun-Wook   Bates, Steven E.   Federico, Laura   Shen, Binghui   O'Connor, Timothy R.  

    Human 3-methyladenine-DNA glycosylase (MPG protein) is involved in the base excision repair (BER) pathway responsible mainly for the repair of small DNA base modifications. It initiates BER by recognizing DNA adducts and cleaving the glycosylic bond leaving an abasic site. Here, we explore several of the factors that could influence excision of adducts recognized by MPG, including sequence context, effect of APE1, and interaction with other proteins. To investigate sequence context, we used 13 different 25bp oligodeoxyribonucleotides containing a unique hypoxanthine residue (Hx) and show that the steady-state specificity of Hx excision by MPG varied by 17-fold. If APE1 protein is used in the reaction for Hx removal by MPG, the steady-state kinetic parameters increase by between fivefold and 27-fold, depending on the oligodeoxyribonucleotide. Since MPG has a role in removing adducts such as 3-methyladenine that block DNA synthesis and there is a potential sequence for proliferating cell nuclear antigen (PCNA) interaction, we hypothesized that MPG protein could interact with PCNA, a protein involved in repair and replication. We demonstrate that PCNA associates with MPG using immunoprecipitation with either purified proteins or whole cell extracts. Moreover, PCNA binds to both APE1 and MPG at different sites, and loading PCNA onto a nicked, closed circular substrate with a unique Hx residue enhances MPG catalyzed excision. These data are consistent with an interaction that facilitates repair by MPG or APE1 by association with PCNA. Thus, PCNA could have a role in short-patch BER as well as in long-patch BER. Overall, the data reported here show how multiple factors contribute to the activity of MPG in cells.
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  • Identification and functional characterization of Histone-like DNA-binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

    Wang, Wenji   Chen, Jianlin   Liao, Baoshan   Xia, Liqun   Hou, Suying   Wang, Zhiwen   Lu, Yishan  

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  • Ligand structures of synthetic deoxa-pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding and inhibitory behaviours

    Xia, Liqun   Lin, Haili   Staniek, Agata   Panjikar, Santosh   Ruppert, Martin   Hilgers, Petra   Williardt, J?rg   Rajendran, Chitra   Wang, Meitian   Warzecha, Heribert   J?ger, Volker   St?ckigt, Joachim  

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