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

    Title Crystal structure of acetoacetate decarboxylase (ADC) (YP_094708.1) from Legionella pneumophila subsp. pneumophila str. Philadelphia 1 at 1.60 A resolution. To be published
    Site JCSG
    PDB Id 3c8w Target Id 376561
    Molecular Characteristics
    Source Legionella pneumophila subsp. pneumophila str. philadelphia 1
    Alias Ids TPS1684,YP_094708.1, PF06314 Molecular Weight 28338.32 Da.
    Residues 254 Isoelectric Point 5.43
    Sequence mdkylsanslegvidnefsmpaprwlntypagpyrfinreffiiayetdpdllqailppdmellepvvk fefirmpdstgfgdytesgqvvpvrykgeeggftismfldchapiaggreiwgfpkklakpklfveedt ligilkygsidiaiatmgykhrpldaekvlesvkkpvfllknipnvdgtplvnqltktyltditvkgaw tgpgslelhphalapisnlyikkivsvshfitdltlpygkvvadyla
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 4
    Resolution (Å) 1.60 Rfree 0.228
    Matthews' coefficent 2.58 Rfactor 0.193
    Waters 711 Solvent Content 52.24

    Ligand Information


    Google Scholar output for 3c8w
    1. Structural classification of proteins and structural genomics: new insights into protein folding and evolution
    A Andreeva, AG Murzin - Acta Crystallographica Section F: Structural , 2010 - scripts.iucr.org

    Protein Summary

     Acetoacatetate decarboxylase (AADase) has long been well accepted as a classic example to illustrate how microenvironment variation can affect enzymes’ activity in vivo. Westheimer and co-workers first hypothesized that a sequencspatial premixing <place w:st="on">Lys</place> 116 should be an essential to provide the large pKa perturbation (-4.5 log units) to enhance the nucleophile on the catalytic Lys115 during the catalysis.  In 1996, Highbarger and Gerlt investigated and proved this hypothesis via a series of mutagenesis and enzymatic studies.   Herein, we present the first crystal structure of AADase from Legionella pneumophila subsp. pneumophila str. <place w:st="on"><city w:st="on">philadelphia</city></place> 1. at the resolution of 1.6 Å.  Very surprisingly, we could not find any structural evidence that the sequence adjacent Lys 116 should be the catalytic obligation to provide pKa disturbing effect to the ε-ammonium of <place w:st="on">Lys</place> 115.  In fact, the side chains from Lys115 and <place w:st="on">Lys</place> 116 orient to a totally opposite direction with an approximate distance of 14.7.  This catalytic <place w:st="on">Lys</place> 115 actually sits in bottom of a very hydrophobic and proton-isolated cavity in AADase.  An unexpected reaction intermediate from bacteria’ nature processing was observed and modeled in the highly conserved active site with a clear covalent double bond distance connecting to the ζ-N of Lys 115, which is supported by both of omitting map and electron density map with 1.6 Å resolution unambiguously.  This modeling of intermediate analogue shows that highly structural and sequence conserved Pro 113 plays a key role in active site construction, pKa disturbing and intermediate stabilization.  This structure also provides the insight to disclose the Glu 86 as the possible general acid/base during the reaction.  This crystal structure of AADase also carries a novel fold mainly dominated by a seven-stranded cone-shaped ß-barrel with that encloses a ~27Å long channel in the active site with closing end. The calculation of interface interaction predicts that a rarely seen quaternary dodecameric structure consisting of a quasi-spherical tetrahedron as the biomolecule.


    The crystal structure of YP_094708.1(Fig. 1A) was determined to 1.6 Å resolution using the MAD method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes four monomers (residues 5–254 for each subunit) in each asymmetric unit and one citrate ions from crystallization is modeled in each subunit. There are total 711 water molecules in each asymmetric unit.  No electron density was observed for the first four residues of the N-terminals in each subunit.  The Matthews’ coefficient (Vm) 24 for PS06314 is 2.58 Å3/Da, and the estimated solvent content is 52.24%. The Ramachandran plot produced by MolProbity 25 shows that 95.3% and 100% of the residues are in favored and allowed regions, respectively.

    Each YP_094708.1 monomer is composed of three α-helices, one 3-10 helix and 17 antiparallel-strands [Fig. 1A] as a single domain.  The total helical content and strand content are 10.8% and 53.8%, respectively. The overall structure of PS06314 molecule should belong to the class of mainly beta-protein in a “sandwich” architecture.  The calculation of interface interaction [PISA] suggest the probable oligomeric association should be a stable dodecamer [Figure 1B, 1C] in solution with the total buried surface area of 65620Å2 according to the crystal lattice packing and assembly analysis. A architecture diagram is shown in Figure 1(c). Twelve identical subunits form a ball-shaped dodacamer in solution, with approximate dimensions of 112.0 Å x 113.8 Å x 99.9 Å (Figure 1(b)).  The main folding core of this protein consists of seven β-strands and two α-helices and makes it folded into a  β-barrel structure as a six-stranded barrel.  A architecture diagram is shown in Figure 1(C). 




    A Novel fold



    Protein YP_094708.1 has been annotated as acetoactate decarboxylase based on its sequence alignment from PSCA (Protein Sequence Comparitive Analysis).  The crystal structure of this enzyme exhibits a novel fold with known function annotation as acetoactate decarboxylase, which mainly consists of β-strands (16 β-strands and 4 α-helics).  The  central folding core (residues 32-47, 66-92, 100-127, 161-177, 194-209 and 232-247) of PS06314 is dominated by seven-stranded β-barrel in a β-cone shape and immediately flanked between a four-stranded and a three-stranded sheets.  The twisting strands of this central core makes these two β-sheet oriented perpendicular to the β-strands of the central β-cone. The conserved active site Y/FPKK locates at the bottom of the central β-cone’s holly core.  Based on the structural architecture [Figure 2A] and the secondary structure of PS06314 [Figure 2B].  A structural similarity search, performed with the coordinates of PS06314 using the DALI, PISA and FFAS server, (reference) only sharing some distant similarity with the structure of Aspartate α-decarboxylase (PDB ID: 1UHD) with an RMSD of 3.39Å over 63 aligned residues with only 10% sequence identity in a “Jelly Rolls” topology. 


    Herein, protein YP_094708.1 should be considered as the first representative of acetoacetate decarboxylase Pfam family to resolve with a novel fold, which is involved in the pathway of Mevalonate Biosynthesis(ref). 2(B)].  The information presented here, in combination with further biochemical and biophysical studies, should yield valuable insights into the functional role of this enzyme for general decarboxylation mechanism.


      YP_094708.1 should be the first structure in Pfam family PF06314 with a novel fold. The Dali search results can not suggest any homolog to YP_094708.1 with considerable similarity.  The SSM search also can not provide any structural homolog. In this structure, Lys125 and Lys126 should be the conserved residue for the catalytic mechanism(1,2,3).


    In this structural, the ?-amine of Lys 125 has a covalent bonding with acetoacetate-like unknown ligand via the formation of a Schiff base intermediate with clear electron density support in the model. The interface interaction calculation suggests the Biomolecule of YP_094708.1 is a dodecamer.


    YP_094708.1 carries a novel fold with known function. More details will be added soon.


    A  B  C  D
    Figure Legend: (A) Ribbon diagram. (B) Dodecamer (C,D) Density at the Schiff base intermediate



    Ligand Summary

    Citrate Ion

    Schiff-Base Intermediate at Lys 125





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