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1z82

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

    Title Crystal structure of glycerol-3-phosphate dehydrogenase (TM0378) from Thermotoga maritima at 2.00 A resolution. To be Published
    Site JCSG
    PDB Id 1z82 Target Id 354146
    Molecular Characteristics
    Source Thermotoga maritima msb8
    Alias Ids TPS1323,TM0378 Molecular Weight 36707.62 Da.
    Residues 329 Isoelectric Point 6.20
    Sequence memrffvlgagswgtvfaqmlhengeevilwarrkeivdlinvshtspyveeskitvratndleeikke dilviaipvqyirehllrlpvkpsmvlnlskgieiktgkrvseiveeilgcpyavlsgpshaeevakkl ptavtlagenskelqkristeyfrvytcedvvgveiagalknviaiaagildgfggwdnakaaletrgi yeiarfgmffgadqktfmglagigdlmvtcnsrysrnrrfgeliargfnplkllessnqvvegaftvka vmkiakenkidmpiseevyrvvyegkpplqsmrdlmrrslkdefwasvvdssl
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 2
    Resolution (Å) 2.00 Rfree 0.211
    Matthews' coefficent 2.03 Rfactor 0.166
    Waters 323 Solvent Content 39.04

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    Ligand Information
    Ligands
    Metals

    Jmol

     
    Google Scholar output for 1z82
    1. The Buccaneer software for automated model building. 1. Tracing protein chains
    K Cowtan - Acta Crystallographica Section D: Biological , 2006 - scripts.iucr.org
     
    2. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard
    TC Terwilliger, PD Adams, RJ Read - Section D: Biological , 2009 - scripts.iucr.org
     

    Protein Summary

    The gene TM0378 from Thermotoga maritima encodes  the enzyme glycerol-3-phosphate dehydrogenase (GlpD) PF01210 PF07479 COG0240.  The enzyme contains two well-defined structural domains with different functions.  The N-terminal domain is the NAD(P)-binding Rossmann-fold domain SCOP51734, while the C-terminal domain is the 6-phosphogluconate dehydrogenase C-terminal domain SCOP48178.  In bacteria, GlpD is a membrane-associated  enzyme from glycolitic pathway, essential for respiration and metabolism.  The structures of the enzyme homologues from other organisms have been solved: 1N1ELeishmania mexicana; 2QCUEscherichia coli.  The determination of the structure of E.Coli GlpD in complex with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide suggests that bacterial GlpD shuttles electrons directly into the respiratory pathway [Ref].  GlpD is considered an attractive drug target because of the dependence of certain pathogenic trypanosomatids on glycolysis for ATP production [Ref]

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    TM0378: A Confirmed glycerol-3-phosphate dehydrogenase with an interesting cofactor specificity

     

    The Thermotoga maritima ORF TM0378 (PDB ID 1Z82) was annotated as a putative glycerol-3-phosphate dehydrogenase (G3PDH) based on sequence and structural comparisons.  TM0378 is similar, in terms of amino acid sequence, to homologs such as gluconate 5-dehydrogenase (Ga5DH) from Archaeglobus fulgidus (PDB ID 1TXG), Leishmania mexicana (PDB ID 1N1E), and Trypanosoma brucei.  Based on initial bioinformatic results, TM0378 was anticipated to have dehydrogenase activity with a preference for NADH as the redox cofactor.  However, experimentally we found that TM0378, while being able to utilize both NADH and NADPH, showed a preference for NADPH for the reduction of dihydroxyacetone phosphate (DHAP); this is reflected in the Michaelis-Menten parameters which we calculated for TM0378.  Although most G3PDHs that have been enzymatically characterized to date are strictly NADH-dependent (or show a preference for NADH over NADPH), comparisons of the Tma sequence to homologs such as the A. fulgidus G3PDH suggest a preference for NADPH over NADH [1].

     

    Bioinformatically, BLAST results revealed that TM0378 shares 30% and 34% sequence identities with the A. fulgidus and L. mexicana homologs, respectively.  Furthermore, DALI results indicate high structural similarity between TM0378 and these two homologs.  Specifically, two structurally conserved domains are found: the N-terminal Rossmann fold and NAD(P)+ motif, and a C-terminal domain from the NAD–dependent G3PDH superfamily. The TM0378 Rossmann fold exhibits the characteristic parallel β-sheet of β-α-β-α-β architecture and the GXGXXG sequence motif.

     

    Enzymatically, Michaelis-Menten kinetics are consistent with annotation of TM0378 as a G3PDH, and also highlight its preference for NADPH over NADH. The kcat for TM0378 with NADPH is about ten-fold greater than with NADH; furthermore, the catalytic efficiency is about three-fold greater (Figure 1 and Table 1).

     

    Cofactor

    Vmax (mM/s)

    KM (mM)

    kcat (s-1)

    kcat/KM (s-1 M-1)

    NADH

    0.0001

    0.86

    0.0219

    25.6

    NADPH

    0.001

    2.70

    0.211

    77.9

     

    Table 1. Comparison of Kinetic Parameters of TM0378 using both NADH and NADPH.Kinetic parameters were calculated using the Hanes-Woolf plots in Figure 1.

    Most G3PDHs exclusively use NADH as a cofactor, and are inactive when only NADPH is available [1].  The unusual preference of A. fulgidus G3PDH for NADPH has been attributed to its R49 residue (see Figure 2),which is moderately well-conserved across multiple species.  The guanidino group of this residue can engage in electrostatic interactions with the 2'-phosphate moiety of NADPH (which is absent in NADH), and possibly with the 5'-phosphate of the adenosine of NADPH [2,3].  Because of the high structural and sequence similarities between the G3PDHs of A. fulgidus and T. maritima, TM0378's preference for NADPH can be elucidated in an analogous manner.

     

    BioLEd Contributers: Kanishk Jain, Ryan Oliver, S. Angel, D. Bezwada, C. Chen, H. Cox, S. Elkin, J. King, C. Modlin, R. Mukherji, Cameron Mura, Carol Price, Linda Columbus.

    Funded by NSF DUE 1044858.

     

    References:

    1.      Sakasegawa S, Hagemeier CH, Thauer RK, Essen LO, Shima S: Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference. Protein Sci 2004, 13(12):3161-3171.

    2.      Charron C, Talfournier F, Isupov MN, Littlechild JA, Branlant G, Vitoux B, Aubury A: The crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 angstrom resolution. J Mol Biol 2000, 297(2):481-500.

    3.      Ermler U, Hagemeier CH, Roth A, Demmer U, Grabarse W, Warkentin E, Vorholt JA: Structure of methylene-tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1. Structure 2002, 10(8):1127-1137.

    Ligand Summary



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    Files (2)

    FileSizeDateAttached by 
     HW_Both_Substrates_kj_v1_Fig1_final.png
    Figure 1. Hanes-Woolf Plot of Substrate Specificity
    256.97 kB15:10, 13 Aug 2012kj3kvActions
     TM0378_Seqalignment_kj_Fig2_final.png
    Figure 2. Tma sequence alignment
    395.45 kB15:10, 13 Aug 2012kj3kvActions
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