Publications

• Computational evidence for the catalytic mechanism of glutaminyl cyclase. A DFT investigation.

  Calvaresi M., Garavelli M., Bottoni A.

  The results of a DFT theoretical investigation on the catalytic mechanism of the QC enzyme are presented. A rather large model-system is used. It includes the most important residues that are believed to play a key-role in the catalysis. The computational results show that the rate-determining ste ... >> More

  The results of a DFT theoretical investigation on the catalytic mechanism of the QC enzyme are presented. A rather large model-system is used. It includes the most important residues that are believed to play a key-role in the catalysis. The computational results show that the rate-determining step of the catalytic process is not the nucleophilic attack leading to the cycle formation (a very easy and fast process with a negligible barrier of 0.8 kcal mol(-1)), but a proton transfer, which is assisted by the Glu201 residue acting as a proton shuttle (general base and general acid). A complex network of hydrogen bonds (involving Asp248 and other residues) contribute to lower the activation barrier for the proton shift which affords the formation of an ammonia molecule bonded to the substrate. The ammonia molecule is a good leaving group which is easily expelled from the substrate in the last step of the catalytic cycle, but remains anchored to the enzyme as a ligand of the zinc cation. The metal plays a key-role in assisting the nucleophilic attack (electrostatic catalysis) since it polarizes the substrate gamma-amide carbonyl group (its electrophilic character increases). Also, the strength of the nucleophilic nitrogen (substrate alpha-amino group) is enhanced by hydrogen bonds involving the Glu201 residue. The computations outline the important role of Trp329 in helping the substrate binding process and stabilizing the cyclization transition state. << Less

  Proteins 73:527-538(2008) [PubMed] [EuropePMC]

• Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

  Huang K.F., Liaw S.S., Huang W.L., Chia C.Y., Lo Y.C., Chen Y.L., Wang A.H.

  Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of ... >> More

  Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC. << Less

  J. Biol. Chem. 286:12439-12449(2011) [PubMed] [EuropePMC]

• Isolation of an isoenzyme of human glutaminyl cyclase: retention in the Golgi complex suggests involvement in the protein maturation machinery.

  Cynis H., Rahfeld J.U., Stephan A., Kehlen A., Koch B., Wermann M., Demuth H.U., Schilling S.

  Mammalian glutaminyl cyclase isoenzymes (isoQCs) were identified. The analysis of the primary structure of human isoQC (h-isoQC) revealed conservation of the zinc-binding motif of the human QC (hQC). In contrast to hQC, h-isoQC carries an N-terminal signal anchor. The cDNAs of human and murine iso ... >> More

  Mammalian glutaminyl cyclase isoenzymes (isoQCs) were identified. The analysis of the primary structure of human isoQC (h-isoQC) revealed conservation of the zinc-binding motif of the human QC (hQC). In contrast to hQC, h-isoQC carries an N-terminal signal anchor. The cDNAs of human and murine isoQCs were isolated and h-isoQC, lacking the N-terminal signal anchor and the short cytosolic tail, was expressed as a fusion protein in Escherichia coli. h-isoQC exhibits 10fold lower activity compared to hQC. Similar to hQC, h-isoQC was competitively inhibited by imidazoles and cysteamines. Inactivation by metal chelators suggests a conserved metal-dependent catalytic mechanism of both isoenzymes. A comparison of the expression pattern of m-isoQC and murine QC revealed ubiquitous expression of both enzymes. However, murine QC transcript formation was higher in neuronal tissue, whereas the amount of m-isoQC transcripts did not vary significantly between different organs. h-isoQC was exclusively localized within the Golgi complex, obviously retained by the N-terminus. Similar resident enzymes of the Golgi complex are the glycosyltransferases. Golgi apparatus retention implies a "housekeeping" protein maturation machinery conducting glycosylation and pyroglutamyl formation. For these enzymes, apparently similar strategies evolved to retain the proteins in the Golgi complex. << Less

  J. Mol. Biol. 379:966-980(2008) [PubMed] [EuropePMC]

• Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation.

  Huang K.-F., Liu Y.-L., Cheng W.-J., Ko T.-P., Wang A.H.-J.

  N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The aberrant formation of pGlu may be related to several pathological processes, such as osteoporosis and amyloidotic diseases. This N-terminal cycliz ... >> More

  N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The aberrant formation of pGlu may be related to several pathological processes, such as osteoporosis and amyloidotic diseases. This N-terminal cyclization reaction, once thought to proceed spontaneously, is greatly facilitated by the enzyme glutaminyl cyclase (QC). To probe this important but poorly understood modification, we present here the structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors. The structure reveals an alpha/beta scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The relatively closed active site displays alternate conformations due to the different indole orientations of Trp-207, resulting in two substrate (glutamine t-butyl ester)-binding modes. The single zinc ion in the active site is coordinated to three conserved residues and one water molecule, which is replaced by an imidazole nitrogen upon binding of the inhibitors. Together with structural and kinetic analyses of several active-site-mutant enzymes, a catalysis mechanism of the formation of protein N-terminal pGlu is proposed. Our results provide a structural basis for the rational design of inhibitors against QC-associated disorders. << Less

  Proc. Natl. Acad. Sci. U.S.A. 102:13117-13122(2005) [PubMed] [EuropePMC]

• An enzyme(s) that converts glutaminyl-peptides into pyroglutamyl-peptides. Presence in pituitary, brain, adrenal medulla, and lymphocytes.

  Busby W.H. Jr., Quackenbush G.E., Humm J., Youngblood W.W., Kizer J.S.

  The mechanism for the post-translational conversion of glutamine to pyroglutamic acid on the N terminus of newly synthesized peptides and proteins is unknown. An assay is reported that permits measurement of the rate of conversion of Gln-His-Pro-NH2 to pyroGlu-His-Pro-NH2 (TRH). Using this assay, ... >> More

  The mechanism for the post-translational conversion of glutamine to pyroglutamic acid on the N terminus of newly synthesized peptides and proteins is unknown. An assay is reported that permits measurement of the rate of conversion of Gln-His-Pro-NH2 to pyroGlu-His-Pro-NH2 (TRH). Using this assay, we demonstrate that the spontaneous cyclization of the N-terminal glutamine of this peptide occurs only slowly under physiological conditions. Furthermore, we describe the presence in rat brain, porcine pituitary, and human B lymphocytes of an enzyme(s) which converts Gln-His-Pro-NH2 into pyroGlu-His-Pro-NH2. The enzyme(s) appears to be a glycoprotein, is maximally active at neutral pH, has a Mr of 55,000, and contains catalytically significant sulfhydryl groups. The product of the enzymatic reaction was confirmed by high resolution fast atom bombardment-mass spectrometry. In preliminary studies, we find that over 90% of the enzyme in bovine adrenal medulla is contained in the soluble chromaffin vesicle fraction. These findings indicate that in vivo the post-translational conversion of a glutaminyl-peptide into a pyroglutamyl-peptide is neither spontaneous nor abiotic as has been previously proposed. << Less

  J Biol Chem 262:8532-8536(1987) [PubMed] [EuropePMC]

• A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis.

  Huang K.F., Wang Y.R., Chang E.C., Chou T.L., Wang A.H.

  QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid art ... >> More

  QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser(160), Glu(201), Asp(248), Asp(305) and His(319)), within which Glu(201) and Asp(248) were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu(201)...Asp(305) and Asp(248)...Asp(305), reduced the steady-state catalysis dramatically. The roles of these two COOH...COOH bonds on catalysis could be partly replaced by COOH...water bonds, but not by COOH...CONH(2) bonds, reminiscent of the low-barrier Asp...Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp(305), a residue located at the centre of the H-bond network, raised the K(m) value of the enzyme by 4.4-19-fold, but decreased the k(cat) value by 79-2842-fold, indicating that Asp(305) primarily plays a catalytic role. In addition, results from mutational studies on Ser(160) and His(319) suggest that these two residues might help to stabilize the conformations of Asp(248) and Asp(305) respectively. These data allow us to propose an essential proton transfer between Glu(201), Asp(305) and Asp(248) during the catalysis by animal QCs. << Less

  Biochem. J. 411:181-190(2008) [PubMed] [EuropePMC]

• Papaya glutamine cyclotransferase shows a singular five-fold beta-propeller architecture that suggests a novel reaction mechanism.

  Guevara T., Mallorqui-Fernandez N., Garcia-Castellanos R., Garcia-Pique S., Ebert Petersen G., Lauritzen C., Pedersen J., Arnau J., Gomis-Ruth F.X., Sola M.

  Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. ... >> More

  Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. The reaction is catalysed by ubiquitous glutaminyl cyclotransferases (QCs), which present two distinct prototypes. Mammalian QCs are zinc-dependent enzymes with an alpha/beta-hydrolase fold. Here we present the 1.6-A-resolution structure of the other prototype, the plant analogue from Carica papaya (PQC). The hatbox-shaped molecule consists of an unusual five-fold beta-propeller traversed by a central channel, a topology that has hitherto been described only for some sugar-binding proteins and an extracellular nucleotidase. The high resistance of the enzyme to denaturation and proteolytic degradation is explained by its architecture, which is uniquely stabilised by a series of tethering elements that confer rigidity. Strikingly, the N-terminus of PQC specifically interacts with residues around the entrance to the central channel of a symmetry-related molecule, suggesting that this location is the putative active site. Cyclisation would follow a novel general-acid/base working mechanism, pivoting around a strictly conserved glutamate. This study provides a lead structure not only for plant QC orthologues, but also for bacteria, including potential human pathogens causing diphtheria, plague and malaria. << Less

  Biol Chem 387:1479-1486(2006) [PubMed] [EuropePMC]

• Isolation and characterization of the glutaminyl cyclases from Solanum tuberosum and Arabidopsis thaliana: implications for physiological functions.

  Schilling S., Stenzel I., von Bohlen A., Wermann M., Schulz K., Demuth H.-U., Wasternack C.

  Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression o ... >> More

  Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris, two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 10(5)-to 10(6)-fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed. << Less

  Biol. Chem. 388:145-153(2007) [PubMed] [EuropePMC]

• Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides.

  Fischer W.H., Spiess J.

  Extracts from bovine pituitary were found to contain an activity catalyzing the conversion of glutaminyl peptides such as [Gln1]gonadotropin-releasing hormone, [Gln1, Gly4]thyrotropin-releasing hormone (H-Gln-His-Pro-Gly-OH), and H-Gln-Tyr-Ala-OH to the respective pyroglutamyl peptides. The TRH pr ... >> More

  Extracts from bovine pituitary were found to contain an activity catalyzing the conversion of glutaminyl peptides such as [Gln1]gonadotropin-releasing hormone, [Gln1, Gly4]thyrotropin-releasing hormone (H-Gln-His-Pro-Gly-OH), and H-Gln-Tyr-Ala-OH to the respective pyroglutamyl peptides. The TRH precursor fragment H-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-OH and the D-glutaminyl stereoisomer of H-Gln-Tyr-Ala-OH did not react under the same conditions. The conversion products were identified by Edman degradation, amino acid analysis, and reversed-phase HPLC. That this activity was exhibited by an enzyme, glutaminyl cyclase, was concluded from the protein character of the activity (revealed by its abolition with trypsin and heat), the Michaelis-Menten relationship between substrate concentration and conversion rate, and the substrate specificity. It was determined that glutaminyl cyclase had a molecular weight of 43,000-50,000, a pH optimum at pH 8, and Km and Vmax values in the range of 60-130 microM and 390-690 pmol/microgram per hr, respectively. Glutaminyl cyclase was not observed to require ATP and could be inhibited with 1.0 M ammonium chloride, which increased the Km and decreased the Vmax value. The subcellular distribution of glutaminyl cyclase corresponded to the one of peptidylglycine alpha-amidating monooxygenase believed to catalyze C-terminal amidations during posttranslational precursor processing. It was also observed that the formation of pyroglutamyl from glutaminyl peptides occurred nonenzymatically; however, the enzymatic reaction carried out with crude extract was found to be approximately 70 times faster than the nonenzymatic reaction enhanced by phosphate. It is speculated that glutaminyl cyclase may participate in the posttranslational processing of hormonal precursors to pyroglutamyl peptides. << Less

  Proc Natl Acad Sci U S A 84:3628-3632(1987) [PubMed] [EuropePMC]