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The Open Protein Structure Annotation Network
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3g7q

    Table of contents
    1. 1. Protein Summary
    2. 2. Ligand Summary

    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
    PDB Id 3g7q Target Id 391498
    Molecular Characteristics
    Source Salmonella typhimurium lt2
    Alias Ids TPS20897,NP_462565.1, 3.40.640.10, 325186 Molecular Weight 46582.42 Da.
    Residues 416 Isoelectric Point 5.74
    Sequence mtfslfgdkftrhsgitrlmedlndglrtpgaimlgggnpahipamqdyfqtlltdmvesgkaadalcn ydgpqgktallnalavllretlgwdiepqnialtngsqsaffylfnlfagrradgstkkvlfplapeyi gyadsgleddlfvsarpniellpegqfkyhvdfehlhigeetgmicvsrptnptgnvitdeelmkldrl anqhniplvidnaygvpfpgiifsearplwnpniilcmslsklglpgsrcgiiiandktitaianmngi islapggmgpammcemikrndllrlsetvikpfyyqrvqqtiaiirrylseerclihkpegaiflwlwf kdlpittellyqrlkargvlmvpghyffpgldkpwphthqcmrmnyvpepdkieagvkilaeeierawreg
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 1
    Resolution (Å) 1.80 Rfree 0.19577
    Matthews' coefficent 2.94 Rfactor 0.17797
    Waters 280 Solvent Content 58.23

    Ligand Information
    Ligands
    Metals

    Jmol

     

    Protein Summary

    NP_462565.1 encodes a protein with 416 residues with a conserved domain belong to AAT_I superfamily. Both NCI blast sequence alignment and Dali search results indicate this protein as an aminotranferase. Strong structural similarity to 1B5O and 1X0M is shown by FFAS and Dali, respectively. JCSG also solved a close homolog from Psychrobacter arcticum 273 http://www.rcsb.org/pdb/explore.do?structureId=3IF2. There is only one subunit of NP_462565.1 in each asymmetric unit. The biomolecule of this target is suggested a dimer by PISA.

    391498_1.png

     

    Figure 1. Protein NP_462565.1 carries a single domain belong to the AAT_I superfamily.

     

    391498_2.png

     

    Figure 2. The biomolecule of NP_462565.1 is a dimer suggested by PISA.

     

     

    391498_3.png

     

     

     

    Figure 3.   Protein NP_462565.1(green) is structuraly similar to 1B5O (Yellow) and 1X0M(Magenta).
     

     

    Annotation by Piotr Kozbial:

    This protein is annotated as valine-pyruvate aminotransferase AvtA by KEGG and is similar to aspartate aminotransferases and their active site cleft, as shown below for PDB:1ix7 (source SuMO server):

      AvtA 1IX7 - MAE Deformation (+ coef.) Deviation (Å) Depth difference Weight
    delta_minus backbone GLY 38 A backbone GLY 38 A 7.9% (3.42) 0.435 0.060 0.6
    aromatic #2 TYR 137 A penta#2 TRP 140 A 11% (5.29) 0.468 0.020 0.9
    amide   ASN 190 A   ASN 194 A 6.9% (8.01) 0.205 0.008 0.75
    delta_minus   TYR 221 A   TYR 225 A 9.6% (3.99) 0.734 0.013 0.6
    aromatic #1 PHE 340 A #1 PHE 360 A 8.7% (5.77) 0.300 0.003 0.9
    delta_plus h12 ARG 388 A h22 ARG 386 A 15% (4.10) 1.104 0.002 0.6

     

    Similarly AvtA is similar to aspartate aminotransferase PDB:1asl (source SuMO server).

     

      AvtA 1ASL - AMA Deformation (+ coef.) Deviation (Å) Depth difference Weight
    amide   ASN 190 A   ASN 194 A 8.2% (8.51) 0.609 0.024 0.75
    delta_minus   TYR 221 A   TYR 225 A 11% (4.52) 0.884 0.033 0.6
    hydroxyl   TYR 221 A   TYR 225 A 11% (4.90) 0.884 0.033 0.65
    aromatic #1 PHE 340 A #2 PHE 360 A 8.1% (6.66) 0.094 0.012 0.9
    delta_plus h12 ARG 388 A h12 ARG 386 A 15% (4.05) 1.361 0.022 0.6
    guanidinium   ARG 388 A   ARG 386 A 5.7% (5.20) 0.221 0.011 0.85

     

    In addition to residues from cleft listed above, there is much smaller adjucent cleft with Cys257, Asp104 and Gly282.

    AvtA is a member of COG3977 (source NCBI) and belongs to:
    (a) 'valine, leucine and isoleucine biosynthesis pathway' (source KEGG database and STRING server),
    (b)inter-pathway connection between 'valine, leucine and isoleucine degradation' and 'valine, leucine and isoleucine biosynthesis'  (source KEGG database and STRING server),
    (c) alanine biosynthesis I pathway (source BioCyc/EcoCyc Pathways and STRING server).

    End of Piotr Kozbial's annotation.

     

    References,

    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   
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    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 the avtA 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.

     

    StAvtA enzymatic 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.

    Ligand Summary

    Reviews

    References

     

    No references found.

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