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3gwq

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    {{ template.Protein{ leadContact:"", title:"Crystal structure of D-serine deaminase from Burkholderia xenovorans LB400 (YP_556991.1) from BURKHOLDERIA XENOVORANS LB400 at 2.00 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-04-01", targetid:"381087", pdbid:"3gwq", source:"Burkholderia xenovorans lb400", relatedPDBs:[], refids:"YP_556991.1, BIG_836, 86760", molwt:"47105.53", residues:"425", isopoint:"5.44", sequence:"mkvtnyqgatidpyskglgmvpgtsiqltdaarlewnllnedvslpaavlyadrvehnlkwmqafvaey gvklaphgkttmapqlfrrqletgawgitlatahqvraayhggvsrvlmanqlvgrrnmmmvaellsdp efeffclvdsvegveqlgeffksvnkqlqvllelgvpggrtgvrdaaqrnavleaitrypdtlklagve lyegvlkeehevreflqsavavtrelveqerfarapavlsgagsawydvvaeefvkasetgkvevvlrp gcylthdvgiyrkaqtdifarnpvakkmgegllpalqlwayvqsipepdraiiglgkrdsafdagmpep arhyrpgneaprdiaasegweifglmdqhaylripagadlkvgdmiafdishpcltfdkwrqvlvvdpa yrvtevietff", method:"XRAY", numchains:"2", resolution:"2.00", rfree:"0.205", mattcoeff:"2.46", rfactor:"0.163", waters:"472", solcontent:"50.10", ligands:"", metals:"", model:"False", uniprot:"Q145Q0", pubmed:"" } }}

    ...

    **************************************************************************************************************************

    Kinetic assays establish 3GWQ as a D-serine deaminase

    The gene for protein YP_556991.1 from Burkholderia xenovorans was putatively annotated by the JCSG as a D-serine deaminase (BxDSD; PDB id 3GWQ; 4.3.1.18). D-serine deaminases catalytically degrade D-serine to pyruvate and ammonia in a mechanism that involves a PLP-aminoacrylate intermediate1. The crystal structure for BxDSD was resolved to 2.00Å by the JCSG.

    Sequence analysis indicates that BxDSD is a member of the fold type-III pyridoxal 5-phosphate (PLP)-dependent enzyme bacterial cryptic D-serine dehydratase family 2,3. This family is characterized by an α/β(TIM) barrel at the N-terminus and a β-sandwich domain at the C-terminus. A catalytic lysine (Lys78 in BxDSD), essential in forming the Schiff base linkage with the C4’ atom in PLP, is conserved across these homologous proteins.

    Kinetics data substantiate the previously putative annotation of BxDSDas a D-serine deaminase. Kinetic parameters were determined using a lactate dehydrogenase (LDH) coupled assay. LDH catalyzes the conversion of pyruvate to lactate with the concomitant oxidation of NADH to NAD+; the concentration of NADH can be monitored spectrophotometrically at 340 nm. Assays were performed at room temperature (approximately 22°C, unless otherwise indicated) in reaction mixtures containing 50 mM potassium phosphate buffer (pH 7.6, unless otherwise indicated), 100 μM PLP, 0.3 mM β-NADH, 6 units/mL LDH from bovine heart, and 0.10 μM BxDSD. 

    A comparison of the kinetic parameters between BxDSD,E. coli DSD (EcDSD) and G. gallus DSD (GgDSD) indicated that BxDSD, fold type III, has similar Km and kcat to GgDSD, fold type III, but a catalytic efficiency much closer to EcDSD, fold type II (Table 1; Figure 1, panel A). A glycine inhibition assay indicated that glycine competitively inhibits BxDSD activity (Figure 1, panel B) (Ki 0.61 mM). 

    BioLEd Contributors: Katherine DeSilva, Zenad Jarrar, Austin McCalla, Elleansar Okwei, Madeline Sargent, Madeline, Jennifer Yi, Catrina Campbell, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

    ...

    References

    1.    Yamada, T., Komoto, J., Takata, Y., Ogawa, H., Pitot, H. C., Takusagawa, F. 2003. Crystal Structure of Serine Dehydratase from Rat Liver. Biochemistry42 (44):12854–65.

    2.    Altshul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. 1990. Basic local alignment search tool. J. Mol. Biol . 215:403-410.

    3.    Madej T, Gibrat JF,Bryant SH. 1995. Threading a database of protein cores. Protein Struct. Funct. Genet. 23:356-369.

    4.    Ito, T., Koga, K., Hemmi, H., Yoshimura, T. 2011. Role of zinc ion for catalytic activity in Dserine dehydratase from Saccharomyces cerevisiae. The FEBS Journal. 279:612-624.

    5.    Suzuki, C.; Murakami, M.; Yokobori, H.; Tanaka, H.; Ishida, T.; Horiike, K.; Nagata, Y. 2011. Rapid determination of free D-serine with chicken D-serine dehydratase. J. of Chromatography B. 879:3326-3330.

    ...

    Table 1. Comparison of kinetic parameters of D-serine substrate.Comparison of kinetic parameters between BxDSD: EcDSD, and GgDSD.

     

    Km mM)

    kcat(s-1)

    kcat/Km (mM-1s-1)

    BxDSD

    0.26

    0.24

    0.92

    EcDSD4

    30

    30

    1.0

    GgDSD5

    0.13

    0.81

    6.2

    ...


    Other changes:

    1. /body/p[11]/span/@style: "font-size: medium;""font-size: medium; line-height: 1.25;"

    Version from 23:28, 8 May 2012

    This revision modified by Admin (Ban)
    {{ template.Protein{ leadContact:"", title:"Crystal structure of D-serine deaminase from Burkholderia xenovorans LB400 (YP_556991.1) from BURKHOLDERIA XENOVORANS LB400 at 2.00 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-04-01", targetid:"381087", pdbid:"3gwq", source:"Burkholderia xenovorans lb400", relatedPDBs:[], refids:"YP_556991.1, BIG_836, 86760", molwt:"47105.53", residues:"425", isopoint:"5.44", sequence:"mkvtnyqgatidpyskglgmvpgtsiqltdaarlewnllnedvslpaavlyadrvehnlkwmqafvaey gvklaphgkttmapqlfrrqletgawgitlatahqvraayhggvsrvlmanqlvgrrnmmmvaellsdp efeffclvdsvegveqlgeffksvnkqlqvllelgvpggrtgvrdaaqrnavleaitrypdtlklagve lyegvlkeehevreflqsavavtrelveqerfarapavlsgagsawydvvaeefvkasetgkvevvlrp gcylthdvgiyrkaqtdifarnpvakkmgegllpalqlwayvqsipepdraiiglgkrdsafdagmpep arhyrpgneaprdiaasegweifglmdqhaylripagadlkvgdmiafdishpcltfdkwrqvlvvdpa yrvtevietff", method:"XRAY", numchains:"2", resolution:"2.00", rfree:"0.205", mattcoeff:"2.46", rfactor:"0.163", waters:"472", solcontent:"50.10", ligands:"", metals:"", model:"False", uniprot:"Q145Q0", pubmed:"" } }}

    ...


    Current version

    This revision modified by cawprice (Ban)
    {{ template.Protein{ leadContact:"", title:"Crystal structure of D-serine deaminase from Burkholderia xenovorans LB400 (YP_556991.1) from BURKHOLDERIA XENOVORANS LB400 at 2.00 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-04-01", targetid:"381087", pdbid:"3gwq", source:"Burkholderia xenovorans lb400", relatedPDBs:[], refids:"YP_556991.1, BIG_836, 86760", molwt:"47105.53", residues:"425", isopoint:"5.44", sequence:"mkvtnyqgatidpyskglgmvpgtsiqltdaarlewnllnedvslpaavlyadrvehnlkwmqafvaey gvklaphgkttmapqlfrrqletgawgitlatahqvraayhggvsrvlmanqlvgrrnmmmvaellsdp efeffclvdsvegveqlgeffksvnkqlqvllelgvpggrtgvrdaaqrnavleaitrypdtlklagve lyegvlkeehevreflqsavavtrelveqerfarapavlsgagsawydvvaeefvkasetgkvevvlrp gcylthdvgiyrkaqtdifarnpvakkmgegllpalqlwayvqsipepdraiiglgkrdsafdagmpep arhyrpgneaprdiaasegweifglmdqhaylripagadlkvgdmiafdishpcltfdkwrqvlvvdpa yrvtevietff", method:"XRAY", numchains:"2", resolution:"2.00", rfree:"0.205", mattcoeff:"2.46", rfactor:"0.163", waters:"472", solcontent:"50.10", ligands:"", metals:"", model:"False", uniprot:"Q145Q0", pubmed:"" } }}

    ...

    **************************************************************************************************************************

    Kinetic assays establish 3GWQ as a D-serine deaminase

    The gene for protein YP_556991.1 from Burkholderia xenovorans was putatively annotated by the JCSG as a D-serine deaminase (BxDSD; PDB id 3GWQ; 4.3.1.18). D-serine deaminases catalytically degrade D-serine to pyruvate and ammonia in a mechanism that involves a PLP-aminoacrylate intermediate1. The crystal structure for BxDSD was resolved to 2.00Å by the JCSG.

    Sequence analysis indicates that BxDSD is a member of the fold type-III pyridoxal 5-phosphate (PLP)-dependent enzyme bacterial cryptic D-serine dehydratase family 2,3. This family is characterized by an α/β(TIM) barrel at the N-terminus and a β-sandwich domain at the C-terminus. A catalytic lysine (Lys78 in BxDSD), essential in forming the Schiff base linkage with the C4’ atom in PLP, is conserved across these homologous proteins.

    Kinetics data substantiate the previously putative annotation of BxDSDas a D-serine deaminase. Kinetic parameters were determined using a lactate dehydrogenase (LDH) coupled assay. LDH catalyzes the conversion of pyruvate to lactate with the concomitant oxidation of NADH to NAD+; the concentration of NADH can be monitored spectrophotometrically at 340 nm. Assays were performed at room temperature (approximately 22°C, unless otherwise indicated) in reaction mixtures containing 50 mM potassium phosphate buffer (pH 7.6, unless otherwise indicated), 100 μM PLP, 0.3 mM β-NADH, 6 units/mL LDH from bovine heart, and 0.10 μM BxDSD. 

    A comparison of the kinetic parameters between BxDSD,E. coli DSD (EcDSD) and G. gallus DSD (GgDSD) indicated that BxDSD, fold type III, has similar Km and kcat to GgDSD, fold type III, but a catalytic efficiency much closer to EcDSD, fold type II (Table 1; Figure 1, panel A). A glycine inhibition assay indicated that glycine competitively inhibits BxDSD activity (Figure 1, panel B) (Ki 0.61 mM). 

    BioLEd Contributors: Katherine DeSilva, Zenad Jarrar, Austin McCalla, Elleansar Okwei, Madeline Sargent, Madeline, Jennifer Yi, Catrina Campbell, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

    ...

    References

    1.    Yamada, T., Komoto, J., Takata, Y., Ogawa, H., Pitot, H. C., Takusagawa, F. 2003. Crystal Structure of Serine Dehydratase from Rat Liver. Biochemistry42 (44):12854–65.

    2.    Altshul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. 1990. Basic local alignment search tool. J. Mol. Biol . 215:403-410.

    3.    Madej T, Gibrat JF,Bryant SH. 1995. Threading a database of protein cores. Protein Struct. Funct. Genet. 23:356-369.

    4.    Ito, T., Koga, K., Hemmi, H., Yoshimura, T. 2011. Role of zinc ion for catalytic activity in Dserine dehydratase from Saccharomyces cerevisiae. The FEBS Journal. 279:612-624.

    5.    Suzuki, C.; Murakami, M.; Yokobori, H.; Tanaka, H.; Ishida, T.; Horiike, K.; Nagata, Y. 2011. Rapid determination of free D-serine with chicken D-serine dehydratase. J. of Chromatography B. 879:3326-3330.

    ...

    Table 1. Comparison of kinetic parameters of D-serine substrate.Comparison of kinetic parameters between BxDSD: EcDSD, and GgDSD.

     

    Km mM)

    kcat(s-1)

    kcat/Km (mM-1s-1)

    BxDSD

    0.26

    0.24

    0.92

    EcDSD4

    30

    30

    1.0

    GgDSD5

    0.13

    0.81

    6.2

    ...


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