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

    Title Crystal structure of NAD-dependent malic enzyme (TM0542) from Thermotoga maritima at 2.61 A resolution. To be published
    Site JCSG
    PDB Id 1vl6 Target Id 282415
    Molecular Characteristics
    Source Thermotoga maritima msb8
    Alias Ids TPS1211,TM0542, 84943 Molecular Weight 41041.13 Da.
    Residues 376 Isoelectric Point 5.32
    Sequence mdaleihrflkgkirtalpvekvdretlsllytpgvadvaracaedpektyvytsrwntvavvsdgsav lglgnigpygalpvmegkaflfkafadidafpiclseseeekiisivkslepsfgginledigapkcfr ilqrlseemnipvfhddqqgtavvvsaaflnalkltekkieevkvvvngigaagynivkflldlgvknv vavdrkgilnendpetclneyhleiaritnperlsgdletalegadffigvsrgnilkpewikkmsrkp vifalanpvpeidpelareagafivatgrsdhpnqvnnllafpgimkgavekrskitknmllsaveaia rscepeperiipeafdmkvhlnvytavkgsa
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 1
    Resolution (Å) 2.61 Rfree 0.23929
    Matthews' coefficent 2.85 Rfactor 0.19189
    Waters 44 Solvent Content 56.87

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    Ligand Information


    Google Scholar output for 1vl6
    1. The Buccaneer software for automated model building. 1. Tracing protein chains
    K Cowtan - Acta Crystallographica Section D: Biological , 2006 - scripts.iucr.org

    Protein Summary

    Malate oxidoreductase (malic enzyme, TM0542) from Thermotoga maritima catalyzes the oxidative decarboxylation of L-malate to pyruvate with the concomitant reduction of the cofactor NAD+ or NADP+. N-terminal domian belongs to Pfam00390, while C-terminal domain to Pfam03949).

    TM0542 has two structural domains: N-terminal domain with aminoacid dehydrogenase-like fold (SCOP sunid:53222) core: 3 layers: ?/?/?; parallel ?-sheet of 4 strands; this domain is decorated with additional structures in family of malic enzyme N-terminal domain (SCOP sunid:53240). C-terminal domain has NAD(P)-binding Rossmann-fold (SCOP sunid:51734) core: 3 layers, ?/?/?; parallel ?-sheet of 6 strands, order 321456. C-terminal domain belongs to aminoacid dehydrogenase-like family (SCOP sunid:51883) with extra N-terminal helix displacing the C-terminal helix (following strand 6) from its usual position creating a family nicotineamide-binding site.

    TM0542 has strong similarity to sequence of proteins from COG0281 (SfcA, malic enzymes) and to several structures of know or predicted malic enzymes (i.e.:2haeA, 1ww8A, 2a9fA, 2aw5A, 1efkA, 1llqA, and 1gq2A).


    Functional Assay

    Based on structure and sequence similarities, TM0542 is hypothesized to be an NAD(P)-dependent malic enzyme (ME), which converts (S)-malate to pyruvate. Using a direct assay, TM0542 demonstrated malic enzyme activity (Figure 1 and Table 1). The decrease in activity at concentrations above 5 mM malate suggests substrate inhibition. In terms of turnover, TM0542 did not demonstrate selectivity for cofactor (NAD+ vs. NADP).

    Structural and sequence comparisons indicate that TM0542 is highly similar to other bacterial MEs (PDB IDs: 2DVM, 2HAE), yet there is no kinetic data for these other bacterial enzymes with which to draw a meaningful activity based comparison. TM0542 is also similar to human NAD(P) ME (PDB ID: 1PJ3), which is well-characterized and provides data for comparison to TM0542 (Table 1). The Km values for malate and NAD are 100-fold lower for TM0542 compared to human ME1,2; however, the human ME turnover values and overall catalytic efficiencies are many orders of magnitude higher than that for TM0542, indicating that TM0542 is far less efficient than the human enzyme. However, for direct comparison, the kinetic parameters should be determined with Mg2+ instead of Mn2+ at pH 7.4.

    Table 1: Comparison of TM0542 and human ME kinetic parameters






    Km (mM)



    kcat/Km (mM-1s-1)

    Km (mM)



    kcat/Km (mM-1s-1)

    Km (mM)












    Human ME#









    *50 mM Tris (pH 8.0), 1 mM EDTA and 5 mM MnCl2; #50 mM Tris (pH 7.4) and 10 mM MgCl2, Not determined due to sigmoidal rather than hyperbolic dependence of activity on NADP concentration


    Additional Findings

    Thetm0542 gene is predicted to be part of a three-gene transcriptional unit (Fig 2). The other two genes, tm0540 and tm0541, encode the N- and C-terminal subunits of fumarate hydratase, respectively. Fumarate hydratase converts (S)-malate to fumarate (and the reverse reaction, when required by metabolic flux). In human ME, a fumarate-binding site that acts as an allosteric activator is present at one of the dimeric interfaces. Although TM0542 and human ME share high structural and sequence similarity, the N-terminal dimer interface and fumarate binding site (consisting of Q64, R67, R91) are not conserved. Attempts at identifying a potential binding site through structural alignments and manual inspection of the dimer interface for the binding motif did not yield promising candidates. Thus, there is no structural evidence for a fumarate allosteric site in TM0542.


    BioLEd Contributors: Joseph Breheny, Kanishk Jain, Michael Billet, John Bui, Huy Do, Golda Harris, Jung Woo Hong, Joo Ho Kim, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.


    1 Hung HC, Kuo MW, Chang GG, Liu GY. Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme). Biochem J. 2005 392:39-45.
     2Hsieh JY, Liu GY, Chang GG, Hung HC. Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of glutamine 362. J Biol Chem. 2006 281:23237-45.

    Ligand Summary





    No references found.

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    Files (2)

    FileSizeDateAttached by 
     Figure 1.png
    Michaelis-Menten Kinetics of TM0542
    39.51 kB15:47, 24 Jan 2012kj3kvActions
    Transcriptional Unit
    20.81 kB15:48, 24 Jan 2012kj3kvActions
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