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3if2

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    {{ template.Protein{ leadContact:"", title:"Crystal structure of Putative amino-acid aminotransferase (YP_265399.1) from Psychrobacter arcticum 273-4 at 2.50 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-07-23", targetid:"391142", pdbid:"3if2", source:"Psychrobacter arcticus 273-4", relatedPDBs:[], refids:"YP_265399.1, 3.40.640.10, 91482", molwt:"49025.04", residues:"443", isopoint:"4.93", sequence:"mkfskfgqkftqptgisqlmddlgdalksdqpvnmlgggnpakidavnelfletykalgndndtgkans saiismanysnpqgdsafidalvgffnrhydwnltsenialtngsqnaffylfnlfggafvnehsqdke sksvdksillpltpeyigysdvhvegqhfaavlphidevthdgeegffkyrvdfealenlpalkegrig aiccsrptnptgnvltdeemahlaeiakrydipliidnaygmpfpniiysdahlnwdnntilcfslski glpgmrtgiivadakvieavsamnavvnlaptrfgaaiatplvandrikqlsdneikpfyqkqatlavk llkqalgdyplmihkpegaiflwlwfkdlpistldlyerlkakgtlivpseyffpgvdvsdyqhaheci rmsiaadeqtlidgikvigevvrelydnk", method:"XRAY", numchains:"2", resolution:"2.50", rfree:"0.221", mattcoeff:"2.96", rfactor:"0.178", waters:"203", solcontent:"58.45", ligands:"", metals:"", model:"False", uniprot:"Q4FPU3", pubmed:"" } }}

    ...

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    Kinetic assays demonstrate valine-pyruvate aminotransferase (VPAT) activity of 3IF2

    Protein YP_265399.1 from Psychrobacter arcticus (PDB id 3IF2)was putatively annotated as a valine-pyruvate aminotransferase (PaVPAT; transaminase C; 2.6.1.66). VPATs are members of the aspartate aminotransferase family. This family is characterized by a pyridoxal-5’-phosphate (PLP) dependent fold type I having a conserved catalytic lysine and conserved PLP-binding residues. A Clustal W sequence alignment of 3IF2 with functionally characterized transaminase C from E. coli and a putative VPAT from Salmonella typhimurium highlights the conserved catalytic lysine, residues associated with PLP binding, and residues expected to be involved in binding substrate (Figure 1)1,2.

    Kinetic data substantiates the annotation of PaVPATas a VPAT. Functional assays were conducted in which the activity was coupled to lactate dehydrogenase (LDH). VPAT enzymes convert L-alanine and α-ketoisovalerate (KIV; 3-Methyl-2-oxobutanoate) to pyruvate and valine through a transamination reaction. The pyruvate product is then converted to lactate by LDH with the concomitant oxidation of NADH to NAD+.  The change in NADH concentration is observed spectrophotometrically at 340 nm. Unless otherwise noted, assays were conducted at 22 °C using the following conditions: 100 mM Tris pH 8, 125 μM PLP, 50 mM L-alanine, 5 mM α-ketoisovalerate, 0.28 mM NADH and 4 U/mL LDH3,4.   L-leucine was at 50 mM when tested as a potential inhibitor against varying amounts of both substrates.

    PaVPATfollows Michaelis-Menten kinetics with respect to both L-alanine and KIV (Figure 2). The Km for L-alanine (4.4 mM) is an order of magnitude higher than for KIV (0.28 mM) (Figure 2; Table 1), indicating that PaVPATmay have a higher binding affinity for KIV than L-alanine2. These values are in line with the published parameters for the VPATs from S. typhimurium (3G7Q; alanine Km = 2.2 mM; KIV Km = 0.10 mM)5 and Brevibacterium flavum ATCC 14067 (alanine Km = 1.5 mM; KIV Km = 0.23 mM)3.

    Kinetic assays with PaVPATin the presence of L-leucine showed competitive inhibition with regard to L-alanine (higher  Km  in the presence of L-leucine) and non-competitive inhibition with respect to KIV (lower Km and Vmax in the presence of L-leucine) (Figure 2; Table 1).

    BioLEd Contributors: Sarah Bentley, David Chang, Jonny Coleman, Kanishk Jain, Autrine Loghmanian, John McGuinn, Shaun Moshasha, David Nyenhuis, Briony Strachan, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

    References

    1.    Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. 2007. Clustal W and clustal X version 2.0. Bioinformatics. 23:2947-2948.

    2.    Segel, I.H. 1975. Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Enzyme Kinetics. Wiley-Interscience, New York.

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

    4.    Ambartsumyan, A. A., Bezirdzhyan, Kh. O. 1994. Isolation and preliminary Characterization of Valine: Pyruvate Aminotransferase fromBrevibacterium flavum. Biochemistry Moscow. 59:1021-1026

    5.    BioLEd TOPSAN entry http://www.topsan.org/Proteins/JCSG/3g7q?highlight=3g7q

    ...

    Table 1. Kinetic parameters for PaVPAT (3IF2)a

     

    L-alanine

    α-ketoisovalerate

    Vmax (μM s-1)

           4.2 ± 0.10

            2.5 ± 0.072

    Km (mM)

           4.4 ± 0.25

            0.28 ± .015

    kcat (s-1)

           7.7 ± 0.18

            4.6 ± 0.029

    kcat/Km (M-1s-1)

    1700 ± 100

    16000 ± 860

    aStandard assay conditions: 100 mM Tris pH 8.0, 0.28 mM NADH, 125 µM PLP, 4 U/mL LDH.

    ...

    Version from 00:20, 9 May 2012

    This revision modified by Admin (Ban)
    {{ template.Protein{ leadContact:"", title:"Crystal structure of Putative amino-acid aminotransferase (YP_265399.1) from Psychrobacter arcticum 273-4 at 2.50 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-07-23", targetid:"391142", pdbid:"3if2", source:"Psychrobacter arcticus 273-4", relatedPDBs:[], refids:"YP_265399.1, 3.40.640.10, 91482", molwt:"49025.04", residues:"443", isopoint:"4.93", sequence:"mkfskfgqkftqptgisqlmddlgdalksdqpvnmlgggnpakidavnelfletykalgndndtgkans saiismanysnpqgdsafidalvgffnrhydwnltsenialtngsqnaffylfnlfggafvnehsqdke sksvdksillpltpeyigysdvhvegqhfaavlphidevthdgeegffkyrvdfealenlpalkegrig aiccsrptnptgnvltdeemahlaeiakrydipliidnaygmpfpniiysdahlnwdnntilcfslski glpgmrtgiivadakvieavsamnavvnlaptrfgaaiatplvandrikqlsdneikpfyqkqatlavk llkqalgdyplmihkpegaiflwlwfkdlpistldlyerlkakgtlivpseyffpgvdvsdyqhaheci rmsiaadeqtlidgikvigevvrelydnk", method:"XRAY", numchains:"2", resolution:"2.50", rfree:"0.221", mattcoeff:"2.96", rfactor:"0.178", waters:"203", solcontent:"58.45", ligands:"", metals:"", model:"False", uniprot:"Q4FPU3", pubmed:"" } }}

    ...

    Current version

    This revision modified by cawprice (Ban)
    {{ template.Protein{ leadContact:"", title:"Crystal structure of Putative amino-acid aminotransferase (YP_265399.1) from Psychrobacter arcticum 273-4 at 2.50 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2009-07-23", targetid:"391142", pdbid:"3if2", source:"Psychrobacter arcticus 273-4", relatedPDBs:[], refids:"YP_265399.1, 3.40.640.10, 91482", molwt:"49025.04", residues:"443", isopoint:"4.93", sequence:"mkfskfgqkftqptgisqlmddlgdalksdqpvnmlgggnpakidavnelfletykalgndndtgkans saiismanysnpqgdsafidalvgffnrhydwnltsenialtngsqnaffylfnlfggafvnehsqdke sksvdksillpltpeyigysdvhvegqhfaavlphidevthdgeegffkyrvdfealenlpalkegrig aiccsrptnptgnvltdeemahlaeiakrydipliidnaygmpfpniiysdahlnwdnntilcfslski glpgmrtgiivadakvieavsamnavvnlaptrfgaaiatplvandrikqlsdneikpfyqkqatlavk llkqalgdyplmihkpegaiflwlwfkdlpistldlyerlkakgtlivpseyffpgvdvsdyqhaheci rmsiaadeqtlidgikvigevvrelydnk", method:"XRAY", numchains:"2", resolution:"2.50", rfree:"0.221", mattcoeff:"2.96", rfactor:"0.178", waters:"203", solcontent:"58.45", ligands:"", metals:"", model:"False", uniprot:"Q4FPU3", pubmed:"" } }}

    ...

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

    Kinetic assays demonstrate valine-pyruvate aminotransferase (VPAT) activity of 3IF2

    Protein YP_265399.1 from Psychrobacter arcticus (PDB id 3IF2)was putatively annotated as a valine-pyruvate aminotransferase (PaVPAT; transaminase C; 2.6.1.66). VPATs are members of the aspartate aminotransferase family. This family is characterized by a pyridoxal-5’-phosphate (PLP) dependent fold type I having a conserved catalytic lysine and conserved PLP-binding residues. A Clustal W sequence alignment of 3IF2 with functionally characterized transaminase C from E. coli and a putative VPAT from Salmonella typhimurium highlights the conserved catalytic lysine, residues associated with PLP binding, and residues expected to be involved in binding substrate (Figure 1)1,2.

    Kinetic data substantiates the annotation of PaVPATas a VPAT. Functional assays were conducted in which the activity was coupled to lactate dehydrogenase (LDH). VPAT enzymes convert L-alanine and α-ketoisovalerate (KIV; 3-Methyl-2-oxobutanoate) to pyruvate and valine through a transamination reaction. The pyruvate product is then converted to lactate by LDH with the concomitant oxidation of NADH to NAD+.  The change in NADH concentration is observed spectrophotometrically at 340 nm. Unless otherwise noted, assays were conducted at 22 °C using the following conditions: 100 mM Tris pH 8, 125 μM PLP, 50 mM L-alanine, 5 mM α-ketoisovalerate, 0.28 mM NADH and 4 U/mL LDH3,4.   L-leucine was at 50 mM when tested as a potential inhibitor against varying amounts of both substrates.

    PaVPATfollows Michaelis-Menten kinetics with respect to both L-alanine and KIV (Figure 2). The Km for L-alanine (4.4 mM) is an order of magnitude higher than for KIV (0.28 mM) (Figure 2; Table 1), indicating that PaVPATmay have a higher binding affinity for KIV than L-alanine2. These values are in line with the published parameters for the VPATs from S. typhimurium (3G7Q; alanine Km = 2.2 mM; KIV Km = 0.10 mM)5 and Brevibacterium flavum ATCC 14067 (alanine Km = 1.5 mM; KIV Km = 0.23 mM)3.

    Kinetic assays with PaVPATin the presence of L-leucine showed competitive inhibition with regard to L-alanine (higher  Km  in the presence of L-leucine) and non-competitive inhibition with respect to KIV (lower Km and Vmax in the presence of L-leucine) (Figure 2; Table 1).

    BioLEd Contributors: Sarah Bentley, David Chang, Jonny Coleman, Kanishk Jain, Autrine Loghmanian, John McGuinn, Shaun Moshasha, David Nyenhuis, Briony Strachan, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

    References

    1.    Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. 2007. Clustal W and clustal X version 2.0. Bioinformatics. 23:2947-2948.

    2.    Segel, I.H. 1975. Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Enzyme Kinetics. Wiley-Interscience, New York.

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

    4.    Ambartsumyan, A. A., Bezirdzhyan, Kh. O. 1994. Isolation and preliminary Characterization of Valine: Pyruvate Aminotransferase fromBrevibacterium flavum. Biochemistry Moscow. 59:1021-1026

    5.    BioLEd TOPSAN entry http://www.topsan.org/Proteins/JCSG/3g7q?highlight=3g7q

    ...

    Table 1. Kinetic parameters for PaVPAT (3IF2)a

     

    L-alanine

    α-ketoisovalerate

    Vmax (μM s-1)

           4.2 ± 0.10

            2.5 ± 0.072

    Km (mM)

           4.4 ± 0.25

            0.28 ± .015

    kcat (s-1)

           7.7 ± 0.18

            4.6 ± 0.029

    kcat/Km (M-1s-1)

    1700 ± 100

    16000 ± 860

    aStandard assay conditions: 100 mM Tris pH 8.0, 0.28 mM NADH, 125 µM PLP, 4 U/mL LDH.

    ...

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