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

    Title Crystal structure of an aspartate aminotransferase (YP_354942.1) from Rhodobacter sphaeroides 2.4.1 at 2.15 A resolution. To be published
    Site JCSG
    PDB Id 3nra Target Id 403422
    Molecular Characteristics
    Source Rhodobacter sphaeroides 2.4.1
    Alias Ids TPS30657,YP_354942.1, 3.40.640.10, 332507 Molecular Weight 43542.97 Da.
    Residues 406 Isoelectric Point 5.89
    Sequence msieakfkklgtdnapgqevrqsaaglealirgapiegrpvdfshgdvdaheptpgafdlfsagvqsgg vqayteyrgdlgirdllaprlaaftgapvdardgliitpgtqgalflavaatvargdkvaivqpdyfan rklveffegemvpvqldyvsadetragldltgleeafkagarvflfsnpnnpagvvysaeeigqiaala arygatviadqlysrlryagasythlraeaavdaenvvtimgpskteslsgyrlgvafgsraiiarmek lqaivslraagysqavlrgwfdeapgwmedriarhqairdellhvlrgcegvfartpqagsylfprlpk lavapaefvkilrlqagvvvtpgtefsphtadsvrlnfsqdheaavaaarrivtlveryra
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 2
    Resolution (Å) 2.15 Rfree 0.200
    Matthews' coefficent 2.40 Rfactor 0.153
    Waters 622 Solvent Content 48.76

    Ligand Information


    Google Scholar output for 3nra
    1. Assessment of ligand_binding residue predictions in CASP9
    T Schmidt, J Haas, TG Cassarino - Structure, Function, and , 2011 - Wiley Online Library
    2. Blind prediction of quaternary structures of homo-oligomeric proteins from amino acid sequences based on templates
    M Morita, M Kakuta, K Shimizu, S Nakamura - 2012 - hoajonline.com
    LC Zohner - 2011 - digitalcommons.unl.edu

    Protein Summary

    This is a member of our aminotran_1_2 family (PF00155). Residue R380 is expected to be catalytic. There are already 450 structures for this family, so is of little interest to us.


    This target is a 406-residue protein encoded by the gene RSP_3437 (JCSG target accession code MJ14530B, GenBank accession code YP_354942.1) from Rhodobacter sphaeroides, a photosynthetic purple bacterium.  It is currently annotated as an aspartate aminotransferase (AspAT) belonging to Pfam PF00155, which is a protein family consisting of aminotransferases class I and II.  AspAT catalyzes the conversion of aspartate and alpha-ketoglutarate to oxaloacetate and glutamate:

    Oxaloacetate + glutamate ⇌ aspartate + α-ketoglutarate

    In humans, AspAT levels are often measured in diagnostic blood tests as an indicator of liver health.

    The structure of RSP_3437 was solved by the Se-MAD method to a resolution of 2.15 Angstroms and reveals an alpha/beta fold that has been observed for other aspartate aminotransferases (Figure 1).  The fold can be classified as belonging to the PLP-dependent transferase superfamily in the SCOP classification scheme.  Like other members of this protein familiy, RSP_3437 contains a lysine (residue 252) that is covalently attached to a pyridoxal-5'-phosphate (PLP) via a Schiff-base linkage at the active site.

    Figure 1.  Structure of a monomer of RSP_3437 (a) gradiently colored from the N- (blue) to the C-terminus (red) and (b) colored according to secondary structure (beta-strands (red), helix (blue), loops (yellow)).  Lysine-252, which is covalently attached to a pyridoxal-5'-phosphate (PLP) via a Schiff-base linkage, is shown in stick representation and labeled.

    MJ14530B-gradient.png    MJ14530B-ss.png


    Crystal packing and PISA analyses suggest that the functional oligomeric state of RSP_3437 is a dimer, consistent with other members of this protein family (Figure 2).

    Figure 2.  Oligomeric state of RSP_3437 is a dimer.  Subunits A and B are in orange and blue, respectively.



    A DALI structural neighbor search reveals almost all of the top matches to be aspartate aminotransferases (Table 1), with their structures being very similar (Figure 3).

    Table 1.  Top structural neighbors of RSP_3437 as assessed by DALI.

    1 1j32 45.3 2.0 367 388 22 ASPARTATE AMINOTRANSFERASE
    2 1gde 45.2 2.3 372 388 20 ASPARTATE AMINOTRANSFERASE [Ref]
    3 1gd9 45.1 2.3 372 388 20 ASPARTATE AMINOTRANSFERASE [Ref]
    4 1dju 44.6 2.2 370 375 20 AROMATIC AMINOTRANSFERASE [Ref]
    5 2o0r 44.2 2.3 365 385 22 RV0858C (N-SUCCINYLDIAMINOPIMELATE [Ref]
    6 5bj4 44.1 2.1 359 366 22 PROTEIN (ASPARTATE AMINOTRANSFERASE) [Ref]
    7 1bkg 44.1 2.1 359 382 22 ASPARTATE AMINOTRANSFERASE [Ref]
    8 5bj3 43.9 2.0 357 363 22 PROTEIN (ASPARTATE AMINOTRANSFERASE) [Ref]
    9 1gck 43.9 2.1 359 382 22 ASPARTATE AMINOTRANSFERASE [Ref]
    10 1bjw 43.8 2.3 361 382 22 ASPARTATE AMINOTRANSFERASE


    Figure 3.  Superposition of RSP_3437 (yellow) with top structural neighbors 1j32 (blue), 1gde (red), and 1dju (green).



    Kinetic analysis of the aspartate aminotransferase 3NRA from Rhodobacter sphaeroides


    The structure of the protein encoded by gene RSP_3439 from Rhodobacter sphaeroides (YP_354942.1 ), was determined to 2.15 Å by the JCSG(PDB id 3NRA). The protein was identified as a putative aspartate aminotransferase (RsAAT; based on structural similarity to other aspartate aminotransferases.


    Sequence analysis of RsAAT indicates that it is a member of fold-type I of PLP-dependent enzymes, specifically subfamily I (PF00155) a conserved family that includes aspartate, tyrosine, alanine, phenylalanine and histidinol-phosphate aminotransferases.1 Structural comparison of RsAAT using the DALI server revealed structural similarities with PLP-dependent aspartate aminotransferases and a PLP-dependent aromatic aminotransferase, 1DJU from Pyrococcus horikoshii (PhAAT)2,3. These sequence and structural comparisons indicate that RsAAT belongs to the alpha-division of subfamily I, whose prokaryotic members show broad specificity that includes aromatic amino acids4.


    Aspartate aminotransferases reversibly catalyze the transamination reaction between L-aspartate and 2-oxoglutarate to yield oxaloacetate and L-glutamate through a ping-pong bi-bi mechanism mediated by the coenzyme PLP5-8. In RsAAT, PLP is covalently bound to the Lys252 residue situated in the active site cleft, which is highly homologous across the (PLP)-dependent aspartate aminotransferase superfamily (fold-type I)9.  The other residues present at the active site are Gly109, Thr110, Gln111, Tyr135, Asn189, Asp217, Tyr220, Gly249, Ser251 Arg260, Tyr338, and Tyr7310. These active site residues are conserved amongst a number of similar proteins: 1GDE, 1J32, 1DJU, 1BJW, and 1ASL (PDB ids).


    Kinetics data was obtained spectrophotometrically using malate dehydrogenase (MDH) as a coupling enzyme. Malate dehydrogenase converts the oxaloacetate, produced by the RsAAT, to malate with the concomitant oxidation of NADH to NAD+. The reaction is monitored at 340 nm, where NADH absorbs strongly but NAD+ does not. Reaction mixtures consisted of 100 mM Tris or 20 mM KH2PO4 (both pH 8.0), 0.2 mM NADH, 10 mM 2-oxoglutarate, 15 μM PLP, 3.6 U mL-1 porcine heart MDH, and 2 µM 3NRA protein. L-aspartate, was varied from 0 to 20 mM.


    While a protein concentration dependent reaction rate was observed for the transamination reaction in both phosphate and Tris buffers, enzymatic activity was significantly lower in 20 mM KH2PO4 pH 8.0 when compared to rates in  100 mM Tris pH 8.0 (Figure 1). This is in agreement with published reports that phosphate can interfere with activity11. RsAAT activity follows Michaelis-Menten kinetics with respect to L-aspartate: Km of 2.1 mM and a kcat of 0.016 s-1 (Figure 2). While the Km for RsAAT is in agreement with other aspartate aminotransferase homologues with reported kinetic parameters (5 mM for 1J32; 2.0 mM for 1BJW, 1.9 mM for 1ASL), the kcat for RsAAT is orders of magnitude lower (187 s-1 for 1J32 and 259 s-1 for 1ASL).


    BioLEd Contributors: Nikolas Hayes, Anita Or, Payal Patel, Michael Pokrass, Paul Riehl, Victor Teran, Joseph Tilitsky, Jennifer Tomlinson Kaitlin Bailey, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.



    1. Finn R. D., Mistry J., Tate J., Coggill P., Heger A., Pollington J. E., Gavin, O. L., Gunasekaran, P., Ceric, G., Forslund, K., Holm, L., Sonnhammer, E. L. L., Eddy, S. R., Bateman, A. 2010. The Pfam Protein Families Database. Nucleic Acids Research. 38:211-222.

    2. Holm L., Rosenstrom P. 2010. Dali Server: Conservation Mapping in 3D. Nucleic Acids Research. 38:545-549.

    3. Matsui I, Matsui E, Sakai Y, Kikuchi H, Kawarabayasi Y, Ura H, Kawaguchi S, Kuramitsu S, Harata K. 2000. The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii. J. Biol. Chem. 275:4871–4879.

    4. Jensen, R., Gu, W. 1996. Evolutionary Recruitment of Biochemically Specialized Subdivisions of Family I within Protein Superfamily of Aminotransferases. J. Bacteriology.178(8):2161-2171.

    5. Kameya, M., Arai, H., Ishii, M., and Igarashi, Y. 2010. Purification of three aminotransferases from Hydogenobacter thermophilus TK-6-novel type of alanine or glycine aminotransferase. FEBS Journal.277(8): 1876-85. 

    6. Hayashi, H., Mizuguchi, H., Miyahara, I., Nakajima, Y., Hirotsu, K., Kagmiyama, H. 2003. Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis. J.Biol.Chem. 278:9481-9488.

    7. de la Torre, F., De Santis, L., Suarez, M. F., Crespillo, R., Canovas, F. M. 2006. Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism. Plant Journal. 46:414-425.

    8. Wu, H., Yang, Y., Wang, S., Qiao, J., Xia, Y., Wang, Y., Gao, S. F., Liu, J., Xue, P. Q., Gao, X. W. 2011. Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3. Febs Journal. 278:1345-1357.

    9. Wrenger, C., Mueller, I. B., Schifferdecker, A. J., Jain, R., Jordanova, R., Groves, M. R. 2011 Specific Inhibition of the Aspartate Aminotransferase of Plasmodium falciparum. J.Mol.Biol.;405:956-971.

    10. Yano, T., Kuramitsu, S., Tanase, S., Morino, Y., and Kagamiyama, H. 1992. Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase. Biochemistry. 31: 5878-5887.

    11. Rej, R., Vanderlinde, R. E. 1975. Effects of buffers on aspartate-aminotransferase activity and association of enzyme with pyridoxal-phosphate. Clin Chem. 21(11):1585-1591.

    Ligand Summary




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