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The Open Protein Structure Annotation Network
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3f6t

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

    Title Crystal structure of aspartate aminotransferase (E.C. 2.6.1.1) (YP_194538.1) from Lactobacillus acidophilus NCFM at 2.15 A resolution. To be published
    Site JCSG
    PDB Id 3f6t Target Id 391714
    Molecular Characteristics
    Source Lactobacillus acidophilus ncfm
    Alias Ids TPS18378,YP_194538.1, 3.40.640.10, 91499 Molecular Weight 60493.51 Da.
    Residues 532 Isoelectric Point 5.08
    Sequence mdnseekklealgafeisrkmlalaqkneksniflnagrgnpnwiqtlarlafvrlvqfgvteskltin ngimagyintdgirerlfafldpdkndedkflidavnychtelglnrdkvvaewvngavannypvpdrc lvntekiinyflqelsykdanlaeqtdlfpteggtaaivyafhslaenhllkkgdkiainepiftpylr ipelkdyelvevdlhsyekndweiepneieklkdpsikalivvnptnptskefdtnalnaikqaveknp klmiisdevygafvpnfksiysvvpyntmlvysysklfgctgwrlgvialneknvfddniahldkvelr qlhkryssvvldpdkmkfidrlcadsrsiglyhtaglstpqqimealfsmthlltstnggsddpyidia rklvserydqlhdamqapkdetdtnthyyslidiyrlaekiygkefrdyltnnfeqvdfllklaekngv vlvdgvgfgakpgelrvsqanlptedyaligkqvlellkeyyeefkqnn
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 2
    Resolution (Å) 2.15 Rfree 0.238
    Matthews' coefficent 2.36 Rfactor 0.177
    Waters 571 Solvent Content 47.92

    Ligand Information
    Ligands
    Metals

    Jmol

     
    Google Scholar output for 3f6t

    Protein Summary

    This protein is classified as an aspartate aminotransferase belonging to Pfam PF00155 (aminotransferase class I and II). FFAS shows hits to many other transferases with very high similarity. A sequence search of the Conserved Domain Database indicates that this protein belongs to COG0436 including Aspartate/tyrosine/aromatic aminotransferase that are involved in amino acid transport and metabolism. From a PSI-BLAST search, this protein sequence matches with many bacterial aminotransferases, but the one that is nearest in sequence with a crystal structure (expectation value=1e-09) is an aromatic aminotransferase from P. horikoshiii (1dju.pdb, Ref1).

    It is present as a dimer in the asymmetric unit of the crystal and crystal packing analysis suggests that is is the stable oligomeric form in solution. There is a covalently bound PLP molecule in each protomer  which has formed a Schiff Base with a neighboring lysine residue to form the modified amino acid LLP (red sticks):

    Dimer2.png

    The bound ligand at Lys312 is surrounded by residues Tyr286, Phe202, Asn254, Arg320, Phe289, Arg39, Tyr442, Tyr443, Trp44 and Tyr205:

     

    PutativeActiveSite.png

     

    Interestingly, this LLP site is not freely solvent accessible in the dimer. Also the LC/MS data do not indicate a change in mass. So, it is not clear how this modification has taken place. The LC/MS data would suggest that there is no modification at the time of purification but the presence of the modification in a region that is not obviously solvent accessible suggests that modification may have taken place after protein expression but before dimer formation.

     

    The LLP is shown in the density modified experimental phases (1.0 sigma map):

    LLPdmmap.png

    The region of electron density in which this LLP was modeled is shown in a Fo-Fc (3.0 sigma) map prior to building it in:

    LLPDiffDen.png

    The LLP is shown in the final 2Fo-Fc map (1.0 sigma map) after refinement:

    LLP.png

     

    A search for other proteins of similar structure using EBI's SSM server shows top hits with glutamine aminotransferase from T. thermophilus (1v2d.pdb) and the human kynurenine aminotranferase I (1w7n.pdb) but the Q-scores are < 0.30. Interestingly, the top hit by FFAS is to 3b46.pdb, also a putative yeast kynurenine aminotransferase (seq id, ~19%, r.m.s.d ~2.9A, Ref2).

    A structure-based sequence alignment using CE shows that some of the residues shown above around the LLP site (sequence in blue below) are also conserved in the 3b46.pdb (pink sequence), i.e. Phe202, Tyr205, Asn254, Tyr286, the modified Lys312 also as LLP,  Arg320, Tyr442 and Tyr443:

      USR1:A   25/40    RGNPNWIQTLARLAFVRLVQFGVTESKLTINNGIXAGYINTDGIRERLFAFLDPDKNDED
    USR2:A 65/63 GFFSYSPPQFAIKEAQKALD----------------------------------------


    USR1:A 85/100 KFLIDAVNYCHTELGLNRDKVVAEWVNGAVANNYPVPDRCLVNTEKIINYFLQELSYKDA
    USR2:A 85/83 ----------------------------IPMVNQYSPTRGRPSLINSLIKLYSP----IY


    USR1:A 145/160 NLAEQ-TDLFPTEGGTAAIVYAFHSLAENHLLKKGDKIAINEPIFTPYLRI-PELKDYEL
    USR2:A 113/111 NTELKAENVTVTTGANEGILSCLMGL-----LNAGDEVIVFEPFFDQYIPNIELCGGKVV


    USR1:A 203/218 VEVDL--------------HSYEKNDWEIEPNEIEKLKDPSIKALIVVNPTNPTSKEFDT
    USR2:A 168/166 YVPINPPKELDQRNTRGEEWT-------IDFEQFEKAITSKTKAVIINTPHNPIGKVFTR


    USR1:A 249/264 NALNAIKQAVEKNPKLXIISDEVYGAFVP--NFKSIYSV---VPYNTXLVYSYSXLFGCT
    USR2:A 221/219 EELTTLGNICVKH-NVVIISDEVYEHLYFTDSFTRIATLSPEIGQLTLTVGSAGXSFAAT


    USR1:A 304/319 GWRLGVIALNEKNVFDDNIAHLDKVELRQLHKRYSSVVLDPDKXKFIDRLCADSRSIGLY
    USR2:A 280/278 GWRIGWVLSLNAELL-----------------------------SYAAKAHTRIC-----


    USR1:A 364/379 HTAGLSTPQQIXEALFSXTHLLTSTNGGSDDPYIDIARKLVSERYDQLHDAXQ---APKD
    USR2:A 306/304 --FASPSPLQEACANSINDALKI--------GYFEKMRQEYINKFKIFTSIFDELGLPYT


    USR1:A 421/436 ETDTNTHYYSLIDIYRLAEKIYGKEFRDYLTNNFE-------------QVDFLLKLAEKN
    USR2:A 356/354 A--PEGTYFVLVDFSKVK----------IPEDYPYPEEILNKGKDFRISHWLINELG---


    USR1:A 468/483 GVVLVDGVGFGA----------KPGELRVSQANL--PTEDYALIGKQVLE
    USR2:A 401/399 -----VVAIPPTEFYIKEHEKAAENLLRFAVCKDDAYLENAVERLKLLKD

     

    The figure below show the Lys12 of this protein modified to LLP by interaction with PLP superimposed on the Lys312/LLP residue from the yeast kynurenine aminotransferase mentioned above (3b46.pdb):

    Superimposedon3B46.png

     References:

    1)J Biol Chem. 2000 Feb 18;275(7):4871-9. The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii.

    2)Wogulis M, Chew ER, Donohoue PD, Wilson DK.

    Identification of formyl kynurenine formamidase and kynurenine aminotransferase from Saccharomyces cerevisiae using crystallographic, bioinformatic and biochemical evidence.
    Biochemistry. 2008 Feb 12;47(6):1608-21. Epub 2008 Jan 19.

    Ligand Summary

    Reviews

    References

     

    No references found.

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