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3g7q

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    Comparing version 00:28, 9 May 2012 by Admin with version 16:30, 14 May 2015 by cawprice.
    {{ template.Protein{ leadContact:"", title:"Crystal structure of valine-pyruvate aminotransferase AvtA (NP_462565.1) from Salmonella typhimurium LT2 at 1.80 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-02-10", targetid:"391498", pdbid:"3g7q", source:"Salmonella typhimurium lt2", relatedPDBs:[], refids:"NP_462565.1, 3.40.640.10, 325186", molwt:"46582.42", residues:"416", isopoint:"5.74", sequence:"mtfslfgdkftrhsgitrlmedlndglrtpgaimlgggnpahipamqdyfqtlltdmvesgkaadalcn ydgpqgktallnalavllretlgwdiepqnialtngsqsaffylfnlfagrradgstkkvlfplapeyi gyadsgleddlfvsarpniellpegqfkyhvdfehlhigeetgmicvsrptnptgnvitdeelmkldrl anqhniplvidnaygvpfpgiifsearplwnpniilcmslsklglpgsrcgiiiandktitaianmngi islapggmgpammcemikrndllrlsetvikpfyyqrvqqtiaiirrylseerclihkpegaiflwlwf kdlpittellyqrlkargvlmvpghyffpgldkpwphthqcmrmnyvpepdkieagvkilaeeierawreg", method:"XRAY", numchains:"1", resolution:"1.80", rfree:"0.19577", mattcoeff:"2.94", rfactor:"0.17797", waters:"280", solcontent:"58.23", ligands:"", metals:"", model:"False", uniprot:"Q8ZL86", pubmed:"" } }}

    ...

    1.     Ura, H.,  Nakai, T.,  Kawaguchi, S.I.,  Miyahara, I.,  Hirotsu, K.,  Kuramitsu, S.   (2001) Substrate recognition mechanism of thermophilic dual-substrate enzyme  J.BIOCHEM.(TOKYO)   130: 89-98       
       
    2.     Chon, H.,  Matsumura, H.,  Koga, Y.,  Takano, K.,  Kanaya, S.   (2005) Crystal structure of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3 at 2.20 A resolution  Proteins   61: 685-688       
      ************************************************************************************************************************************************

    Kinetic assays confirm that 3G7Q has valine-pyruvate aminotransferase activity

    ...

    The structure and sequence of AvtA (NP_462565.1) from Salmonella typhimurium (StAvtA; PDB id 3G7Q) was determined to a resolution of 1.61 Å1. On the basis of structural and sequence similarities, 3G7Q was putatively identified as a valine-pyruvate aminotransferase (VPAT; EC 2.6.1.66). Enzymatic analysis using coupled enzyme assays supports the role of StAvtA (3G7Q) as a VPAT.

    ...

    Sequence analysis indicates that StAvtA is a PLP-dependent aspartate aminotransferases2. PLP binding sites and catalytic residues conserved across VPATs are found in StAvtA. One protein with particularly high similarity is E. coli transaminase C, a VPAT encoded by theavtA gene3.  Transaminase C is responsible for several interconversions in amino acid syntheses and is the terminal step in valine biosynthesis3. High levels of L-alanine in the cell repress expression of the avtA gene4.

    ...

    Coupled kinetic assay data support the putative function of StAvtA as a valine-pyruvate aminotransferase. The enzymatic activity of StAvtA was determined in the forward and reverse directions using coupled kinetic assays with lactate dehydrogenase (LDH) or L-alanine dehydrogenase (ALADH) (Figure 1). LDH converts pyruvate, the product of the 3G7Q reverse reaction, to lactate, oxidizing NADH in the process. ALADH converts L-alanine, the product of the 3G7Q forward reaction, to pyruvate, reducing NAD+ in the process. The inter-conversion of NAD+ and NADH allows the coupled assays to be monitored spectrophotometrically at 340 nm.

    ...

    Reverse reaction assays consisted of 40 μM PLP, 0.2 mM NADH, 150 mM Tris pH 8, 5 units/mL LDH. When the alanine concentration was varied, reaction assays included 20 mM 3-methyl-2-oxobutanoate and 0.30 μM StAvtA; when 3-methyl-2-oxobutanoate concentration was varied, reaction assays included 20 mM L-alanine and 0.052 μM StAvtA. Forward reaction assays for the determination of valine parameters consisted of 40 μM PLP, 1 mM pyruvate, 1 mM NAD+, 100 mM Tris pH 8, 0.45 units/mL ALADH, 8 μM StAvtA, and varying L-valine concentrations.

    ...

    StAvtAenzymatic activity followed Michaelis-Menten kinetics in both the forward and reverse directions. The Km for L-alanine was 2.2 mM and the kcat was 3.8 s-1; the Km for 3-methyl-2-oxobutanoate was 0.010 mM and the kcat was 19 s-1 (Figure 2, panels A and B).  These values are similar to published parameters for the VPATs from Psychrobacter arcticus (3IF2; alanine Km = 4.4 mM, kcat = 77.8 s-1; 3-methyl-2-oxobutanoate Km = 0.28 mM, kcat = 46.3 s-1; 100 mM Tris pH 8, 125 µM PLP, 0.28 mM NADH, 4 U/mL LDH, and 54 nM 3IF2)5 and Brevibacterium flavum ATCC 14067 (alanine Km = 1.5 mM; 3-methyl-2-oxobutanoate Km = 0.23 mM)6. The StAvtA Km for L-valine was 0.65 mM and the kcat was 0.0014 s-1 (Figure 2, panel C), indicating that valine may have a higher affinity than alanine but the overall turnover is much slower.

    ...

    BioLEd Contributors: Caleigh Azumaya, Ziyi Gao, Creigh Greensmith, Marc Lipman, Hannah Mabe, Matt Saunders, Briony Strachen, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

    ...

    References

    ...

    1.    Bjellqvist, B., Hughes, G.J., Pasquali, Ch., Paquet, N., Ravier, F., Sanchez, J.-Ch.,Frutiger, S. & Hochstrasser, D.F. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis. 1993, 14:1023-1031.

    2.    Altschul, S., Madden, T., Schaeffer, A., Zhang, J., Zhang, Z., Miller, W., Lipman, D. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.

    3.    Whalen, W., Berg, C. 1982. Analysis of an avtA:Mu d1 (Ap lac) mutant: Metabolic role of transaminase C.. J. Bacteriol. 150:739-746.

    4.    Whalen, W. A., Berg, C. M. 1984. Gratuitous repression of avtA in Escherichia coli and Salmonella typhimurium. J Bacteriol. 158(2):571-574.

    5.    BioLEd TOPSAN entry http://www.topsan.org/Proteins/JCSG/3if2?highlight=3if2

    6.     Ambartsumyan, A. A., Bezirdzhyan, Kh. O. 1994. Catalytic Properties of Valine Pyruvate Aminotransferases from Brevibacterium flavum. Biochemistry Moscow. 59:1027-1032.

    ...


    Other changes:

    1. /body/h4/@class: "topsan h4 topsanProtein" ⇒ nothing

    Version from 00:28, 9 May 2012

    This revision modified by Admin (Ban)
    {{ template.Protein{ leadContact:"", title:"Crystal structure of valine-pyruvate aminotransferase AvtA (NP_462565.1) from Salmonella typhimurium LT2 at 1.80 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-02-10", targetid:"391498", pdbid:"3g7q", source:"Salmonella typhimurium lt2", relatedPDBs:[], refids:"NP_462565.1, 3.40.640.10, 325186", molwt:"46582.42", residues:"416", isopoint:"5.74", sequence:"mtfslfgdkftrhsgitrlmedlndglrtpgaimlgggnpahipamqdyfqtlltdmvesgkaadalcn ydgpqgktallnalavllretlgwdiepqnialtngsqsaffylfnlfagrradgstkkvlfplapeyi gyadsgleddlfvsarpniellpegqfkyhvdfehlhigeetgmicvsrptnptgnvitdeelmkldrl anqhniplvidnaygvpfpgiifsearplwnpniilcmslsklglpgsrcgiiiandktitaianmngi islapggmgpammcemikrndllrlsetvikpfyyqrvqqtiaiirrylseerclihkpegaiflwlwf kdlpittellyqrlkargvlmvpghyffpgldkpwphthqcmrmnyvpepdkieagvkilaeeierawreg", method:"XRAY", numchains:"1", resolution:"1.80", rfree:"0.19577", mattcoeff:"2.94", rfactor:"0.17797", waters:"280", solcontent:"58.23", ligands:"", metals:"", model:"False", uniprot:"Q8ZL86", pubmed:"" } }}

    ...


    Version as of 16:30, 14 May 2015

    This revision modified by cawprice (Ban)
    {{ template.Protein{ leadContact:"", title:"Crystal structure of valine-pyruvate aminotransferase AvtA (NP_462565.1) from Salmonella typhimurium LT2 at 1.80 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-02-10", targetid:"391498", pdbid:"3g7q", source:"Salmonella typhimurium lt2", relatedPDBs:[], refids:"NP_462565.1, 3.40.640.10, 325186", molwt:"46582.42", residues:"416", isopoint:"5.74", sequence:"mtfslfgdkftrhsgitrlmedlndglrtpgaimlgggnpahipamqdyfqtlltdmvesgkaadalcn ydgpqgktallnalavllretlgwdiepqnialtngsqsaffylfnlfagrradgstkkvlfplapeyi gyadsgleddlfvsarpniellpegqfkyhvdfehlhigeetgmicvsrptnptgnvitdeelmkldrl anqhniplvidnaygvpfpgiifsearplwnpniilcmslsklglpgsrcgiiiandktitaianmngi islapggmgpammcemikrndllrlsetvikpfyyqrvqqtiaiirrylseerclihkpegaiflwlwf kdlpittellyqrlkargvlmvpghyffpgldkpwphthqcmrmnyvpepdkieagvkilaeeierawreg", method:"XRAY", numchains:"1", resolution:"1.80", rfree:"0.19577", mattcoeff:"2.94", rfactor:"0.17797", waters:"280", solcontent:"58.23", ligands:"", metals:"", model:"False", uniprot:"Q8ZL86", pubmed:"" } }}

    ...

    1.     Ura, H.,  Nakai, T.,  Kawaguchi, S.I.,  Miyahara, I.,  Hirotsu, K.,  Kuramitsu, S.   (2001) Substrate recognition mechanism of thermophilic dual-substrate enzyme  J.BIOCHEM.(TOKYO)   130: 89-98   
     
    2.     Chon, H.,  Matsumura, H.,  Koga, Y.,  Takano, K.,  Kanaya, S.   (2005) Crystal structure of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3 at 2.20 A resolution  Proteins   61: 685-688   
    ************************************************************************************************************************************************

    Kinetic assays confirm that 3G7Q has valine-pyruvate aminotransferase activity

    ...

    The structure and sequence of AvtA (NP_462565.1) from Salmonella typhimurium (StAvtA; PDB id 3G7Q) was determined to a resolution of 1.61 Å1. On the basis of structural and sequence similarities, 3G7Q was putatively identified as a valine-pyruvate aminotransferase (VPAT; EC 2.6.1.66). Enzymatic analysis using coupled enzyme assays supports the role of StAvtA (3G7Q) as a VPAT.

    ...

    Sequence analysis indicates that StAvtA is a PLP-dependent aspartate aminotransferases2. PLP binding sites and catalytic residues conserved across VPATs are found in StAvtA. One protein with particularly high similarity is E. coli transaminase C, a VPAT encoded by theavtA gene3.  Transaminase C is responsible for several interconversions in amino acid syntheses and is the terminal step in valine biosynthesis3. High levels of L-alanine in the cell repress expression of the avtA gene4.

    ...

    Coupled kinetic assay data support the putative function of StAvtA as a valine-pyruvate aminotransferase. The enzymatic activity of StAvtA was determined in the forward and reverse directions using coupled kinetic assays with lactate dehydrogenase (LDH) or L-alanine dehydrogenase (ALADH) (Figure 1). LDH converts pyruvate, the product of the 3G7Q reverse reaction, to lactate, oxidizing NADH in the process. ALADH converts L-alanine, the product of the 3G7Q forward reaction, to pyruvate, reducing NAD+ in the process. The inter-conversion of NAD+ and NADH allows the coupled assays to be monitored spectrophotometrically at 340 nm.

    ...

    Reverse reaction assays consisted of 40 μM PLP, 0.2 mM NADH, 150 mM Tris pH 8, 5 units/mL LDH. When the alanine concentration was varied, reaction assays included 20 mM 3-methyl-2-oxobutanoate and 0.30 μM StAvtA; when 3-methyl-2-oxobutanoate concentration was varied, reaction assays included 20 mM L-alanine and 0.052 μM StAvtA. Forward reaction assays for the determination of valine parameters consisted of 40 μM PLP, 1 mM pyruvate, 1 mM NAD+, 100 mM Tris pH 8, 0.45 units/mL ALADH, 8 μM StAvtA, and varying L-valine concentrations.

    ...

    StAvtAenzymatic activity followed Michaelis-Menten kinetics in both the forward and reverse directions. The Km for L-alanine was 2.2 mM and the kcat was 3.8 s-1; the Km for 3-methyl-2-oxobutanoate was 0.010 mM and the kcat was 19 s-1 (Figure 2, panels A and B).  These values are similar to published parameters for the VPATs from Psychrobacter arcticus (3IF2; alanine Km = 4.4 mM, kcat = 77.8 s-1; 3-methyl-2-oxobutanoate Km = 0.28 mM, kcat = 46.3 s-1; 100 mM Tris pH 8, 125 µM PLP, 0.28 mM NADH, 4 U/mL LDH, and 54 nM 3IF2)5 and Brevibacterium flavum ATCC 14067 (alanine Km = 1.5 mM; 3-methyl-2-oxobutanoate Km = 0.23 mM)6. The StAvtA Km for L-valine was 0.65 mM and the kcat was 0.0014 s-1 (Figure 2, panel C), indicating that valine may have a higher affinity than alanine but the overall turnover is much slower.

    ...

    BioLEd Contributors: Caleigh Azumaya, Ziyi Gao, Creigh Greensmith, Marc Lipman, Hannah Mabe, Matt Saunders, Briony Strachen, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

    ...

    References

    ...

    1.    Bjellqvist, B., Hughes, G.J., Pasquali, Ch., Paquet, N., Ravier, F., Sanchez, J.-Ch.,Frutiger, S. & Hochstrasser, D.F. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis. 1993, 14:1023-1031.

    2.    Altschul, S., Madden, T., Schaeffer, A., Zhang, J., Zhang, Z., Miller, W., Lipman, D. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.

    3.    Whalen, W., Berg, C. 1982. Analysis of an avtA:Mu d1 (Ap lac) mutant: Metabolic role of transaminase C.. J. Bacteriol. 150:739-746.

    4.    Whalen, W. A., Berg, C. M. 1984. Gratuitous repression of avtA in Escherichia coli and Salmonella typhimurium. J Bacteriol. 158(2):571-574.

    5.    BioLEd TOPSAN entry http://www.topsan.org/Proteins/JCSG/3if2?highlight=3if2

    6.     Ambartsumyan, A. A., Bezirdzhyan, Kh. O. 1994. Catalytic Properties of Valine Pyruvate Aminotransferases from Brevibacterium flavum. Biochemistry Moscow. 59:1027-1032.

    ...


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