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Publikation

Prasad, A.; Breithaupt, C.; Nguyen, D.-A.; Lilie, H.; Ziegler, J.; Stubbs, M. T.; Mechanism of chorismate dehydratase MqnA, the first enzyme of the futalosine pathway, proceeds via substrate-assisted catalysis J. Biol. Chem. 298, 102601, (2022) DOI: 10.1016/j.jbc.2022.102601

MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Details of the MqnA reaction mechanism remain unclear. Here, we present crystal structures of Streptomyces coelicolor MqnA and its active site mutants in complex with chorismate and the product 3-enolpyruvyl-benzoate, produced during heterologous expression in Escherichia coli. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with the enol pyruvyl group of chorismate acting as catalytic base. Surprisingly, structures of the mutant Asn17Asp with copurified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway.
Publikation

Wasternack, C.; Hause, B.; OPDA-Ile – a new JA-Ile-independent signal? Plant Signal Behav. 11, e1253646, (2016) DOI: 10.1080/15592324.2016.1253646

Expression takes place for most of the jasmonic acid (JA)-induced genes in a COI1-dependent manner via perception of its conjugate JA-Ile in the SCFCOI1-JAZ co-receptor complex. There are, however, numerous genes and processes, which are preferentially induced COI1-independently by the precursor of JA, 12-oxo-phytodienoic acid (OPDA). After recent identification of the Ile-conjugate of OPDA, OPDA-Ile, biological activity of this compound could be unequivocally proven in terms of gene expression. Any interference of OPDA, JA, or JA-Ile in OPDA-Ile-induced gene expression could be excluded by using different genetic background. The data suggest individual signaling properties of OPDA-Ile. Future studies for analysis of an SCFCOI1-JAZ co-receptor-independent route of signaling are proposed.
Publikation

Wasternack, C.; Goetz, S.; Hellwege, A.; Forner, S.; Strnad, M.; Hause, B.; Another JA/COI1-independent role of OPDA detected in tomato embryo development Plant Signal Behav. 7, 1349-1353, (2012) DOI: 10.4161/psb.21551

Jasmonates (JAs) are ubiquitously occurring signaling compounds in plants formed in response to biotic and abiotic stress as well as in development. (+)-7-iso-jasmonoyl isoleucine, the bioactive JA, is involved in most JA-dependent processes mediated by the F-box protein COI1 in a proteasome-dependent manner. However, there is an increasing number of examples, where the precursor of JA biosynthesis, cis-(+)-12-oxophytodienoic acid (OPDA) is active in a JA/COI1-independent manner. Here, we discuss those OPDA-dependent processes, thereby giving emphasis on tomato embryo development. Recent data on seed coat-generated OPDA and its role in embryo development is discussed based on biochemical and genetic evidences.
Publikation

Wasternack, C.; Xie, D.; The genuine ligand of a jasmonic acid receptor: Improved analysis of jasmonates is now required Plant Signal Behav. 5, 337-340, (2010) DOI: 10.4161/psb.5.4.11574

Jasmonic acid (JA), its metabolites, such as the methyl ester or amino acid conjugates as well as its precursor 12-oxophytodienoic acid (OPDA) are lipid-derived signals. JA, OPDA and JA-amino acid conjugates are known to function as signals in plant stress responses and development. More recently, formation of JA-amino acid conjugates and high biological activity of JA-Isoleucine (JA-Ile) were found to be essential in JA signalling. A breakthrough was the identification of JAZ proteins which interact with the F-box protein COI1 if JA-Ile is bound. This interaction leads to proteasomal degradation of JAZs being negative regulators of JA-induced transcription. Surprisingly, a distinct stereoisomer of JA-Ile, the (+)-7-iso-JA-Ile ((3R,7S) form) is most active. Coronatine, a bacterial phytotoxine with an identical stereochemistry at the cyclopentanone ring, has a similar bioactivity . This was explained by the recent identification of COI1 as the JA receptor and accords well with molecular modelling studies. Whereas over the last two decades JA was quantified to describe any JA dependent process, now we have to take into account a distinct stereoisomer of JA-Ile. Until recently a quantitative analysis of (+)-7-iso-JA-Ile was missing presumable due to its equilibration to (-)-JA-Ile. Now such an analysis was achieved. These aspects will be discussed based on our new knowledge on JA perception and signalling.
Publikation

Ziegler, J.; Brandt, W.; Geißler, R.; Facchini, P. J.; Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis J. Biol. Chem. 284, 26758-26767, (2009) DOI: 10.1074/jbc.M109.030957

Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel α-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.
Publikation

Schilling, S.; Wasternack, C.; Demuth, H.-U.; Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution Biol. Chem. 389, (2008) DOI: 10.1515/BC.2008.111

Several mammalian peptide hormones and proteins from plant and animal origin contain an N-terminal pyroglutamic acid (pGlu) residue. Frequently, the moiety is important in exerting biological function in either mediating interaction with receptors or stabilizing against N-terminal degradation. Glutaminyl cyclases (QCs) were isolated from different plants and animals catalyzing pGlu formation. The recent resolution of the 3D structures of Carica papaya and human QCs clearly supports different evolutionary origins of the proteins, which is also reflected by different enzymatic mechanisms. The broad substrate specificity is revealed by the heterogeneity of physiological substrates of plant and animal QCs, including cytokines, matrix proteins and pathogenesis-related proteins. Moreover, recent evidence also suggests human QC as a catalyst of pGlu formation at the N-terminus of amyloid peptides, which contribute to Alzheimer's disease. Obviously, owing to its biophysical properties, the function of pGlu in plant and animal proteins is very similar in terms of stabilizing or mediating protein and peptide structure. It is possible that the requirement for catalysis of pGlu formation under physiological conditions may have triggered separate evolution of QCs in plants and animals.
Publikation

Schilling, S.; Stenzel, I.; von Bohlen, A.; Wermann, M.; Schulz, K.; Demuth, H.-U.; Wasternack, C.; Isolation and characterization of the glutaminyl cyclases from Solanum tuberosum and Arabidopsis thaliana: implications for physiological functions Biol. Chem. 388, 145-153, (2007) DOI: 10.1515/BC.2007.016

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 105- to 106-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.
Publikation

Schneider, K.; Kienow, L.; Schmelzer, E.; Colby, T.; Bartsch, M.; Miersch, O.; Wasternack, C.; Kombrink, E.; Stuible, H.-P.; A New Type of Peroxisomal Acyl-Coenzyme A Synthetase from Arabidopsis thaliana Has the Catalytic Capacity to Activate Biosynthetic Precursors of Jasmonic Acid J. Biol. Chem. 280, 13962-13972, (2005) DOI: 10.1074/jbc.M413578200

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function. Because of their high structural and sequence similarity with bona fide 4CLs and their highly hydrophobic putative substrate-binding pockets, the 4CL-like proteins At4g05160 and At5g63380 were selected for detailed analysis. Following heterologous expression, the purified proteins were subjected to a large scale screen to identify their preferred in vitro substrates. This study uncovered a significant activity of At4g05160 with medium-chain fatty acids, medium-chain fatty acids carrying a phenyl substitution, long-chain fatty acids, as well as the jasmonic acid precursors 12-oxo-phytodienoic acid and 3-oxo-2-(2′-pentenyl)-cyclopentane-1-hexanoic acid. The closest homolog of At4g05160, namely At5g63380, showed high activity with long-chain fatty acids and 12-oxo-phytodienoic acid, the latter representing the most efficiently converted substrate. By using fluorescent-tagged variants, we demonstrated that both 4CL-like proteins are targeted to leaf peroxisomes. Collectively, these data demonstrate that At4g05160 and At5g63380 have the capacity to contribute to jasmonic acid biosynthesis by initiating the β-oxidative chain shortening of its precursors.
Publikation

Schilling, S.; Manhart, S.; Hoffmann, T.; Ludwig, H.-H.; Wasternack, C.; Demuth, H.-U.; Substrate Specificity of Glutaminyl Cyclases from Plants and Animals Biol. Chem. 384, 1583-1592, (2003) DOI: 10.1515/BC.2003.175

Glutaminyl cyclases (QC) catalyze the intramolecular cyclization of N-terminal glutamine residues of peptides and proteins. For a comparison of the substrate specificity of human and papaya QC enzymes, a novel continuous assay was established by adapting an existing discontinuous method. Specificity constants (kcat/Km) of dipeptides and dipeptide surrogates were higher for plant QC, whereas the selectivity for oligopeptides was similar for both enzymes. However, only the specificity constants of mammalian QC were dependent on size and composition of the substrates. Specificity constants of both enzymes were equally pH-dependent in the acidic pH-region, revealing a pKa value identical to the pKa of the substrate, suggesting similarities in the substrate conversion mode. Accordingly, both QCs converted the L-?homoglutaminyl residue in the peptide H-?homoGln-Phe-Lys-Arg-Leu-Ala-NH2 and the glutaminyl residues of the branched peptide H-Gln-Lys(Gln)-Arg-Leu-Ala-NH2 as well as the partially cyclized peptide H-Gln-cyclo( N?-Lys-Arg-Pro-Ala-Gly-Phe). In contrast, only QC from C. papaya was able to cyclize a methylated glutamine residue, while this compound did not even inhibit human QC-catalysis, suggesting distinct substrate recognition pattern. The conversion of the potential physiological substrates gastrin, neurotensin and [GlN1]-fertilization promoting peptide indicates that human QC may play a key role in posttranslational modification of most if not all pGlu-containing hormones.
Publikation

Schilling, S.; Niestroj, A. J.; Rahfeld, J.-U.; Hoffmann, T.; Wermann, M.; Zunkel, K.; Wasternack, C.; Demuth, H.-U.; Identification of Human Glutaminyl Cyclase as a Metalloenzyme J. Biol. Chem. 278, 49773-49779, (2003) DOI: 10.1074/jbc.M309077200

Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 ± 1 and 295 ± 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 ± 0.02, 6.93 ± 0.03, and 5.60 ± 0.05 for imidazole, methylimidazole, and benzimidazole, respectively, match the values obtained by titrimetric pKa determination, indicating the requirement for an unprotonated nitrogen for binding to QC. Similarly, the pH dependence of the kinetic parameter Km for the QC-catalyzed conversion of H-Gln-7-ami-no-4-methylcoumarin also implies that only N-terminally unprotonated substrate molecules are bound to the active site of the enzyme, whereas turnover is not affected. The results reveal human QC as a metal-dependent transferase, suggesting that the active site-bound metal is a potential site for interaction with novel, highly potent competitive inhibitors.
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