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==Introduction== ''Drosophila melanogaster'' glutaminyl cyclase (DromeQC; also known as CG10487, CG32412 or Dmel\CG32412) is a globular protein part of the α/β-hydrolase superfamily<ref name="main">PMID: 22897232</ref>. DromeQC is an aminoacyltransferase (EC 2.3.2.5) that acts on N-terminal glutamine or glutamate residues, producing a protease-resistant cap in substrate proteins<ref name="main"/>. The human orthologue of DromeQC (hQC) has been implicated in stabilizing amyloid Aβ peptides involved in neurodegenerative disorders such as Alzheimers<ref name="schilling">PMID: 18836460</ref>. It has been shown that DromeQC has a similar overall fold to hQC, as well as a conserved active site<ref name="main"/>. Thus, DromeQC is an attractive candidate for transgenic models and mechanistic studies, which may further characterize neurodegenerative disorders and help develop treatments. ===DNA--> RNA--> Protein=== DromeQC is encoded by chromosome 3L, locus 64F4-64F5 in the ''D. melanogaster'' genome<ref name="genecard">DromeQC Gene Card. NCBI. [http://www.ncbi.nlm.nih.gov/gene?cmd=Retrieve&dopt=Graphics&list_uids=38663]</ref>. This gene is transcribed into a 1622 nucleotide transcript, containing 5' (36 nucleotides) and 3' (83 nucleotides) untranslated regions <ref name="genecard"/>. The translated protein contains 340 residues corresponding to a mass of 38,028 Da<ref>DromeQC. UniProt. [http://www.uniprot.org/uniprot/Q9VRQ9]</ref>. It also contains a 27 residue signal sequence, suggesting its involvement in the secretory pathway <ref name="schilling"/>. ===Protein Family=== DromeQC belongs to the α/β-hydrolase fold superfamily; this superfamily exhibits a β-sheet core (5-8 strands) connected to α-helices, forming an α/β/α sandwhich<ref name="family">PMID: 23193256</ref>. The α/β-hydrolases are well characterized in the ESTHER database (ESTerases and α/β-Hydrolase Enzymes and Relatives), which as of present contains 168 protein families<ref>Lenfant, N., Hotelier, T., Velluet, E., Bourne, Y., Marchot, P., and A Chatonnet. (2013) ESTHER, the database of the alpha/beta-hydrolase fold superfamily of proteins: tools to explore diversity of functions. Nucleic Acids Research 41: D423-9. [http://bioweb.ensam.inra.fr/ESTHER/general?what=index]</ref>. Most of the proteins in this superfamily function via a conserved catalytic triad - a nucleophile, acid and base - which is present on the loops of the active site<ref name="family"/>. Further information on catalysis is found in the 'Catalytic Mechanism' section on this page. It is interesting that DromeQC prevents protein degradation from aminopeptidases, yet theses two enzymes share a common fold and active site<ref>PMID: 15028118</ref>. This suggests that the enzymes act in a similar manner, with opposing effects on substrate. ==Structure== <Structure load='4F9U' size='350' frame='true' align='right' caption='Figure 1. Various modelling of DromeQC structure, as determined by X-ray crystallography (PDB: 4F9U). Click on the green links within the text to display different models.' scene='Insert optional scene name here'/> ===Visual Walkthrough=== The following descriptions can be viewed in the 3D JSmol image when any of the green links are clicked (Figure 1). DromeQC is a <scene name='58/580851/Two_chains/2'>homodimer</scene> consisting of an <span style="font-size:150%"><font color='blue'>A Chain</font></span> and a <span style="font-size:150%"><font color='limegreen'>B Chain</font></span>. This homodimer can also be viewed in <scene name='58/580851/Trace/1'>trace</scene> and <scene name='58/580851/Space_fill/1'>space filling</scene> representations. The backbone of DromeQC can be easily seen as an <scene name='58/580851/N_to_c_rainbow/1'>N to C rainbow</scene>. Here, polypeptide progression in each chain is depicted by the following template: {{Template:ColorKey_Amino2CarboxyRainbow}} The <scene name='58/580851/Composition/1'>composition</scene> of DromeQC consists of either <span style="font-size:150%">{{Template:ColorKey Composition Protein}}, {{Template:ColorKey Composition Ligand}}</span>, or the <span style="font-size:150%">{{Template:ColorKey Composition Solvent}}</span> in which it was crystallized (water). When DromeQC <scene name='58/580851/Secondary/1'>secondary structure</scene> is colour coated <span style="font-size:150%">({{Template:ColorKey_Helix}}, {{Template:ColorKey_Strand}})</span> the global fold may be visualized nicely. This fold is driven by <scene name='58/580851/Polar/1'>hydrophobic/polar</scene> residues. The majority of <span style="font-size:150%">{{Template:ColorKey_Hydrophobic}}</span> residues lie within the enzyme's interior, consistent with the hydrophobic collapse folding theory. In contrast, most <span style="font-size:150%">{{Template:ColorKey_Polar}}</span> residues reside on the exterior where they contact the polar solvent. Similarly, <scene name='58/580851/Charge/1'>charged residues</scene>, either <span style="font-size:150%">{{Template:ColorKey_Charge_Anionic}}</span> or <span style="font-size:150%">{{Template:ColorKey_Charge_Cationic}}</span>, appear to cluster on the outside of the protein. From analysis of charged residues, it can be seen that a salt bridge may connect the two monomers (Chain A→Chain B: ARG35→GLU64). [[Image:QCtopology.gif|thumb|200px|left|Figure 3. Topology of DromeQC<ref>PDB Sum Entry 4F9U. EMBL-EBI. [https://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=4f9u&template=protein.html&r=wiring&l=1&chain=A]</ref>]] [[Image:Cropped_dimer.jpg|thumb|400px|right|Figure 2. A 3D graphical representation displaying the homodimer glutaminyl cyclase from ''Drosophila melanogaster'' (PDB: 4F9U). Secondary structure is depicted by red (α-helix) and yellow (β-strand) ribbons, glycosyl groups are coloured pink, while hydrogen bonds between the two monomers are shown by dotted green lines. The active site of QC contains a chelated zinc ion represented by a gray sphere. Also bound to the active site of this crystal structure and depicted as blue is the inhibitor 1-(3,4-dimethoxyphenyl)-3-[3-(1H-imidazol-1-yl)propyl]thiourea.]][[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 4. A comparison between the active sites of DromeQC crystalized either with a PBD150 inhibitor (right, PDB: 4F90) or without (left, PDB: 4FWU). Protein loops surrounding the active site are denoted in dark blue, providing a scaffold for a catalytic Zn²⁺ (gray) to be chelated by three residues (light blue).The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]] ===Topology and Overall Structure<ref name="main"/>=== As shown above, DromeQC is made up of 2 identical, independent monomers that come together to form an asymmetric <scene name='58/580851/Two_chains/2'>homodimer</scene> (Figure 1). The subunits are connected via 4 hydrogen bonds (Chain A→Chain B: ARG35 NH2→GLU64 OE2, ARG43 NH2→ASN71 O, ARG43 NH2→PHE75 O, ARG43 NH1→PHE75 O) and surface complementarity (Figure 2). The subunits exhibit a globular α/β-hydrolase fold, characterized by a central twisted β-sheet motif consisting of 5 parallel strands (β1 and β3-β6) and an antiparallel β2 strand (Figures 2&3). This β-center is flanked by 9 surrounding α-helices; 2 fill the concave face (α5, α7), 7 fill the convex face (α1- α5, α8, α9) with one helix at the edge (α6) of each monomer. DromeQC is glycosylated (with up to 7 carbohydrate moieties) at the N42 position. These polysaccharide tags increase the solubility of DromeQC, and appear to have no affect of protein activity<ref name="main"/>. Within each subunit is 1 disulfide bond (C113→C136) linking the β3 strand to the α3 helix. These cysteine residues are situated close to the active site and are conserved in the human orthologue, suggesting a pivotal role in catalysis<ref name="schilling"/>. However, this hypothesis was ruled out when cysteines were replaced with alanines via site-directed mutagenesis; in these experiments no kinetic differences were observed in the DromeQC mutant<ref name="main"/>. In contrast, these mutations did affect structural differences of DromeQC, as determined by thermal unfolding experiments<ref name="main"/>. These results correspond to the structural stabilization of this disulfide bond in hQC, with no apparent effect on kinetic activity<ref>Ruiz-Carrillo, D., Koch, B., Parthier, C., Wermann, M., Dambe, T., Buchholz, M., Ludwig, H., Heiser, U., Rahfeld, J., Stubbs, M. T., Schilling, S., and H. Demuth. (2011) Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center. Biochemistry. 50: 6280-6288. [http://dx.doi.org/10.1021/bi200249h DOI: 10.1021/bi200249h]</ref>. ===Active Site=== The active site of DromeQC is located on four loops that lack secondary structure (Figure 4). Using these loops as a scaffold, a catalytic zinc ion is chelated via D131 OD2, E171 OE2, and H297 NE2. Thus, under the absence of substrate or inhibitor, Zn²⁺ exhibits trivalency (Figure 4). However, when DromeQC was crystalized in presence of a PBD150 inhibitor, Zn²⁺ was additionally chelated by the PBD150 imidazole moiety<ref name="main"/>. In a similar fashion, it is plausible that the γ oxygen of glutamine in peptide substrates chelates Zn²⁺. This would lead to proper position, polarization, and stabilization of substrate for cyclization. ===Inhibitor binding<ref name="main"/>=== Binding of PBD150 to DromeQC utilizes π-π, arene-H, and hydrogen bonding interactions (Figure 4). The dimethoxyphenyl phenyl group of PBD150 is stabilized by π-π interactions with F292. This phenyl group is highly flexibily, however, as only weak electron density was observed during crystal structure analysis<ref name="main"/>. Such flexibility could be essential for substrates to cyclize. Also stabilizing the inhibitor is an arene-H interaction made between the imidazole moiety of PBD150 and W296. The first carbon upstream of this imidazole ring forms an additional arene-H contact with W176. Finally, the sulfur contained in the thiourea group makes a hydrogen bond with D271. This sulfur could mimic a carbonyl oxygen in the backbone of a peptide, suggesting a possible substrate binding mechanism. ==Function== [[Image:Cyclase_reaction.png|thumb|500px|right|Figure 5. Cyclization of a terminal glutamine residue via DromeQC.]] The N-terminus of many proteins (ie gonadotropin releasing hormone and thyrotropin-releasing hormone) contain a pyroglutamic acid (pGlu) residue<ref>PMID: 196172</ref>. A pGlu ‘cap’ protects these proteins against degradation by aminopeptidases, and influences the conformation of the hormone or its associated receptor, leading to their activation<ref name="schilling"/>. Cyclization also leads to decreased basicity in the peptide. Though cyclization of Gln-tRNA to pGlu-tRNA has been shown to occur in papaya latex,<ref>PMID: 4881333</ref> N terminal pGlu formation must be post translational due to an essential methionine that initiates translation in all organisms. ===Catalytic Mechanism=== DromeQC is the enzyme responsible for this post-translational processing of polypeptides. DromeQC catalyzes the cyclization of N-terminal glutamine, and to a lesser extent glutamate, into pyroglutamic acid (5-oxo-L-proline, or <Glu) (Figure 5). This enzyme can be categorized as follows: :-transferase (2) ::-acyltransferase (2.3) :::-aminoacyltransferase (2.3.2) ::::-acts on glutaminyl/glutamyl residues (2.3.2.5) The cyclization reaction occurs via a nucleophilic attack of the α-amine on the γ carbon in the glutamine side chain. The enzymatic mechanism for DromeQC is still undetermined, but it seems plausible that it follows that of its human orthologue. In hQC, the N-terminus of the peptide substrate is inserted into the active site pocket, where the γ amide oxygen chelates the catalytic zinc ion<ref name="mechanism">PMID: 18470930</ref>. This ion-dipole interaction causes carbonyl polarization, making it a better electrophile. To facilitate the reaction, a conserved glutamate (Glu201) acts as both a general base and acid. Glu201 abstracts a proton from the α-amino group, causing it to nucleophilically attack the γ amide oxygen. This produces a tetrahedral intermediate with a charged oxygen that is stabilized by Zn²⁺. Glu201 then protonates the γ amide nitrogen, and an amine group is expelled as the carbonyl reforms. Also essential to this mechanism is a conserved aspartate (Asp248) that coordinates/stabilizes the leaving amine group. ==Localization== DromeQC is known to localize in the brain and peripheral nerves of ''D. melanogaster''<ref name="schilling"/>. This fact was unveiled by the discovery of adipokinetic hormone; this protein has an N-terminal pGlu, supporting a post translational modification via DromeQC<ref>PMID: 2117437</ref>. Adipokinetic hormone was found to localize in neurone and nerve endings by immunohistochemistry, suggesting that it functions as a locally released modulator in tissues such as heart and skeletal muscle<ref>PMID: 6342796</ref>. Thus, It has been suggested that DromeQC is part of the secretory pathway, being targeted to secretory vesicles where hormone maturation takes place<ref name="schilling"/>. This claim is supported by a 27 residue signal sequence contained at the N-terminus of DromeQC<ref name="schilling"/>. Further support stems from immunohistochemical evidence that DromeQC and its modified substrate are excreted from the cell, where they can be found in the extracellular medium<ref name="schilling"/>. ==References== <references/>

• Template:ColorKey Amino2CarboxyRainbow
• Template:ColorKey Charge Anionic
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• Template:ColorKey Composition Protein
• Template:ColorKey Composition Solvent
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