The Open Protein Structure Annotation Network
PDB Keyword


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

    Title Structure of the first representative of Pfam family PF04016 (DUF364) reveals enolase and Rossmann-like folds that combine to form a unique active site with a possible role in heavy-metal chelation. Acta Crystallogr.,Sect.F 66 1167-1173 2010
    Site JCSG
    PDB Id 3l5o Target Id 360984
    Molecular Characteristics
    Source Desulfitobacterium hafniense dcb-2
    Alias Ids TPS1457,DHAF_12NOV03_CONTIG1056_REVISED_GENE1477, PF04016, 382598 Molecular Weight 27742.57 Da.
    Residues 251 Isoelectric Point 5.61
    Sequence mweiydamingipedflvdelvcgtthsvirsgngvglgpnrpfetrmpmltqnllglplrvaagcvks wnyveasiglaainayynnpqvarehgvifsdakrvedrmndpfimsqnevkgkkvgvvghfphlesll epicdlsilewspeegdyplpasefilpecdyvyitcasvvdktlprllelsrnarritlvgpgtplap vlfehglqelsgfmvkdnarafrivagaekvkiysagqkvtikk
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 2
    Resolution (Å) 2.01 Rfree 0.215
    Matthews' coefficent 2.59 Rfactor 0.171
    Waters 238 Solvent Content 52.56

    Ligand Information


    Google Scholar output for 3l5o
    1. Structure of the first representative of Pfam family PF04016 (DUF364) reveals enolase and Rossmann-like folds that combine to form a unique active site with a
    MD Miller, L Aravind, C Bakolitsa, CL Rife - Section F: Structural , 2010 - scripts.iucr.org

    Protein Summary

    The Dhaf4260 gene (uniprot: B8FUJ5) from Desulfitobacterium hafniense encodes a protein of unknown function from a family (DUF364 (PF04016)) found in archaea and bacteria. The structure (3l5o and 2h1q are the same) reveals a two-domain organization with a PLP-dependent transferase-like fold preceded by a N-terminal domain adopting an enolase N-terminal domain-like fold. Structural and genetic context analysis suggests a possible role in the anaerobic biosynthesis of vitamin B12.[Ref]



    Dhaf4260 is a two-domain α+β protein (Fig.1).The N-terminal domain (residues 1-102) adopts an enolase N-terminal-like fold characterized by three helices with up-down topology and a three-stranded anti-parallel β-sheet. The C-terminal domain (residues 110-251) adopts a PLP-dependent transferase-like fold characterized by a three-layer α/β/α sandwich topology with a seven-stranded mixed β-sheet. 




    Fig. 1. Crystal structure of Dhaf4260 from Desulfitobacterium hafniense. (A) Stereo ribbon diagram of the Dhaf4260 monomer color-coded from N-terminus (blue) to C-terminus (red). Helices H1–H13 and β-strands (β1−β10) are indicated. (B) Diagram showing the secondary structure elements of Dhaf4260 superimposed on its primary sequence. The labeling of secondary structure elements is in accord with PDBsum, where α-helices are labeled sequentially (H1, H2, H3 etc.), β-strands are labeled (A, B, C etc.) according to the β-sheets to which they are assigned, β-turns and γ-turns are designated by their respective Greek letters (β, γ), and red loops indicate β-hairpins. For Dhaf4260, the α-helices (H1-H6, H8-H10 and H12), 310-helices (H7, H11 and H13), β-sheets (A-B, comprising strands β1-β3 and β4-β10 respectively), β-turns (β) and β-hairpins are indicated.




    A search with FATCAT shows the strongest structural similarity of Dhaf4260 with precorrin methyltransferases and decarboxylases (PDB ids: 1f38, 2yxd, main-chain rmsd 3.1-3.2 Å over 170-173 residues, sequence identity 9%), enzymes involved in the anaerobic pathway of cobalamin (vitamin B12) biosynthesis (Scott & Roessner, 2002)[Ref], (Keller et al., 2002). The similarity involves both fold and topology, and maps to the C-terminal domain of Dhaf4260 excluding the last two helices (H12 and H13) and strand (β10). An additional helix (H9) inserted in the loop between Dhaf4260 strands β5 and β6 is not found in precorrins (Fig. 2). The N-terminal domain of Dhaf4260 shows strong similarity to the N-terminal domain of enolases (PDB id: 4enl, main-chain rmsd 1.9 Å over 60 residues, sequence identity 8%). Dhaf4260 contains an additional helix (H1) at the N-terminus (Fig. 2).



    Fig. 2. Dhaf4260 exhibits structural similarity to enolases and cobalamin-binding proteins. (A) Top and (B) Side stereo view of the structural superposition of Dhaf4260 (PDB id: 3l5o, residues 1-251, in blue) with the enolase N-terminal domain from Saccharomyces cerevisiae (PDB id: 4enl, residues 1-139, in gray) and precorrin 8W from Methanobacterium thermoautotrophicum (PDB id: 1f38, residues 1-186 in cyan).


    Rather weak Dali top hits using 3l5o as query are: the cystathionine gamma-synthase 1i41 (Z-scr=9), the histidinol-phosphate amino transferase 1gex (Z-scr=8.8), the cystathionine beta-lyase 1cl2 (z-scr=8.0) and 1f38 (Z-scr=7).

    Size exclusion chromatography in combination with static light scattering, indicate that a tetramer is the predominant quaternary form. This is in agreement with the oligomerization state of MT0146, a putative precorrin-8w decarboxylase (Keller et al., 2002), although crystal packing analysis of the Dhaf4260 structure only supports a monomer or dimer. Moreover, the long tetramerization hairpin observed in precorrin is in Dhaf4260 replaced by helices H12 and H13.


    An Asp or Glu residue that interacts with the hydroxyl groups of the ribose is the most conserved feature of adenosyl (e.g. ATP, NAD, S-adenosyl-methionine) binding sites (Carugo and Argos, 1997). Asp62 fulfills this role in MT0146 and is superimposable with E148 that is strictly conserved among Dhaf4260 homologs. We predict it might serve a similar function in Dhaf4260. However, the GGSGG loop implicated in binding S-adenosyl-methionine through an induced-fit mechanism thereby completing the precorrin binding site, is absent from Dhaf4260 suggesting a different mechanism at play for Dhaf4260.


    The enolase superfamily, comprising mandelate racemase (MR), muconate lactonizing enzyme (MLE) and enolases, is a group of functionally related enymes each of which is organized into two domains: a substrate specificity-determining capping N-terminal domain, followed by a TIM-barrel that contains the metal ion ligands and acid/base catalysts at the C-terminal ends of different β-strands (Gerlt and Babbitt, 2001). The dimer interface is conserved among prokaryotes and eykaryotes with dimerization proposed to play a role in promoting subunit stability (Kuhnel and Luisi, 2001).The first two strands of the enolase N-terminal domain β-sheet and the long β3-H1 loop, that connects the third strand to the first helix and closes onto the active site upon substrate binding, form part of the dimerization interface. In Dhaf4260, the dimer interface of the N-terminal domain is located along the interdomain interface with the precorrin-like C-terminal domain, and the β3-H1 loop is much shorter.


    A search with HHPred showed significant homology of the C-terminal domain of Dhaf4260 (residues 135-217) with PF03446 (P-value 9.5E-05 over Dhaf4260 residues 123-204), PF02826 (P-value 7.2E-05 over residues 119-200) and PF00670 (P-value 3.3E-05 over residues 120-200). All three families contain NAD-binding domains, with PF00670 belonging to an S-adenosyl-L-homocysteine hydrolase (AdoHcyase), a vitamin B12-dependent enzyme of the activated methyl cycle. A remote homology with another family of B12-binding proteins (PF02310, P-value 0.00071 over residues 135-217) is also observed.




    DUF364-specific features


    A search in DOMINE, a database of protein domain interactions, reports no combination of enolase N-terminal domain-like fold (PF03952) with S-adenosyl-L-methionine-dependent methyltrasferases (PF08241). To our knowledge, this is the first occurrence of such a domain combination. However, functionally related combinations have been reported, for example of the enolase N-terminal domain with the C-terminal domain of porphobilinogen deaminase (PF03953), an enzyme implicated in the biosynthesis of uroporphyrinogen III, a precursor of vitamin B12.


    Helix H9, one of the additional secondary structure elements not observed in the precorrin structures, lies at the center of a large, positively charged grove along the inter-domain interface (Figs. 3A, 3B). According to ConSurf, this is a highly conserved region of the molecule. Residues contributing to this positive surface include Arg31, Arg42 and Arg47 from the N-terminal domain and Lys223, Arg227, Arg230, Lys237, Lys239, Lys246 from the C-terminal domain. The only solvent-exposed aromatic side-chains in Dhaf4260 are located at the inter-domain interface and involve Trp70 ad Tyr72 from the N-terminal domain, and Trp149 from the C-terminal domain (Figs. 3B).


    A search of this cavity using the graph-matching algorithm Isocleft (Najmanovich et al., 2008) reveals a B12-dependent glutamate mutase as the top hit (PDB id: 1ccw). Other significant hits involved typical (PDB ids: 2nap) and atypical (PDB id: 1q90) hemes, factor F430 (PDB id: 1e6v) and flavohemoglobin (PDB id: 1cqx). Metals (iron-sulfur clusters, divalent cations) and other adenine dinucleosides (bis(adenosine)-5’-pentaphosphate) and dinucleotides (FAD, NAD(P)) also scored highly. The corrin ring (four pyrrole subunits) that comprises the core of vitamin B12, is similar to porphyrin, found in hemes, but with one of the bridging methylene groups removed. Uroporphyrinogen III, is an intermediate in the biosynthesis of vitamin B12 but also of heme, siroheme, chlorophylls and factor F430 (Scott & Roessner, 2002). Hence, the ligands predicted for Dhaf4260 share both biochemical and structural similarity with vitamin B12.




    Fig. 3. Electrostatic surface properties of Dhaf4260. (A) Electrostatic surface potential distribution of Dhaf4260. Positive potential is in blue (3kT), negative is in red (-3kT). (B) Ribbon representation of Dhaf4260 in the same orientation as in (A). Charged residues at the inter-domain interface and residues with solvent-exposed aromatic side-chains are indicated.




    The genomic neighborhood of DUF2478 (PF10649) homologs shows a high degree of confidence in a predicted functional association with a number of proteins involved in transport and synthesis of rare metals such as iron (WS1133), tungstate (MTH926), vanadium (RPA1384, RPA1385) and molybdate (MTH924, Mbar_A1307, amb0153), as well as several domains (e.g. radical SAM domain protein (Mlab_0336), cobyrinic acid a,c-diamide synthase (Mlab_0341), cobalamin biosynthesis CbiM precursor (Pisl_0217), cobalamin biosynthesis protein N (MTH928)) implicated in vitamin synthesis. Metal transporters, such as TonB, have been shown to be able to complement deficiencies in the acquisition of vitamin B12 (Poole et al., 1996) while recently it was shown that different nutrient and metabolite capture proteins can share the same energy-coupling module (Rodionov et al., 2009).


    Two pathways to adenosylcobalamin corrin ring formation have been described – aerobic and anaerobic – that differ in the point of cobalt insertion (Roth et al., 1996). The aerobic pathway to adenosylcobalamin evolved around 2 billion years ago, whereas the anaerobic route dates to approximately 4 billion years (Santander et al., 1997). Given the presence of DUF364 homologs in archaea, the oldest life form, we propose that this family is involved in the anaerobic pathway leading to vitamin B12 biosynthesis. The inter-domain cleft with the added extensions (absent from precorrin structures) could act as a binding site for vitamin B12 or a precursor and, by analogy with the N-terminal enolase-like fold, the Dhaf4260 N-terminal domain could be implicated in stereospecific catalysis.


    Availability of more DUF364 member sequences and structures might shed light on the evolutionary history of this intriguing protein family.  

    Ligand Summary








    1. (No Results)


      Discuss this publication
    2. (No Results)


      Discuss this publication
    Tag page
    Viewing 1 of 1 comments: view all
    Various people in the world receive the <a href="http://lowest-rate-loans.com/topics/business-loans">business loans</a> in different banks, just because that is simple and comfortable.
    Posted 21:18, 27 Jun 2010
    Viewing 1 of 1 comments: view all
    You must login to post a comment.
    All content on this site is licensed under a Creative Commons Attribution 3.0 License
    Powered by MindTouch