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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

Bürstenbinder, K.; Savchenko, T.; Müller, J.; Adamson, A. W.; Stamm, G.; Kwong, R.; Zipp, B. J.; Dinesh, D. C.; Abel, S.; Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1 J. Biol. Chem. 288, 1871-1882, (2013) DOI: 10.1074/jbc.M112.396200

Calcium (Ca2+) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (Kd ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca2+/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.
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

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

Gidda, S. K.; Miersch, O.; Levitin, A.; Schmidt, J.; Wasternack, C.; Varin, L.; Biochemical and Molecular Characterization of a Hydroxyjasmonate Sulfotransferase from Arabidopsis thaliana J. Biol. Chem. 278, 17895-17900, (2003) DOI: 10.1074/jbc.M211943200

12-Hydroxyjasmonate, also known as tuberonic acid, was first isolated from Solanum tuberosum and was shown to have tuber-inducing properties. It is derived from the ubiquitously occurring jasmonic acid, an important signaling molecule mediating diverse developmental processes and plant defense responses. We report here that the gene AtST2a from Arabidopsis thaliana encodes a hydroxyjasmonate sulfotransferase. The recombinant AtST2a protein was found to exhibit strict specificity for 11- and 12-hydroxyjasmonate with Km values of 50 and 10 μm, respectively. Furthermore, 12-hydroxyjasmonate and its sulfonated derivative are shown to be naturally occurring inA. thaliana. The exogenous application of methyljasmonate to A. thaliana plants led to increased levels of both metabolites, whereas treatment with 12-hydroxyjasmonate led to increased level of 12-hydroxyjasmonate sulfate without affecting the endogenous level of jasmonic acid. AtST2a expression was found to be induced following treatment with methyljasmonate and 12-hydroxyjasmonate. In contrast, the expression of the methyljasmonate-responsive gene Thi2.1, a marker gene in plant defense responses, is not induced upon treatment with 12-hydroxyjasmonate indicating the existence of independent signaling pathways responding to jasmonic acid and 12-hydroxyjasmonic acid. Taken together, the results suggest that the hydroxylation and sulfonation reactions might be components of a pathway that inactivates excess jasmonic acid in plants. Alternatively, the function of AtST2a might be to control the biological activity of 12-hydroxyjasmonic acid.
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.
Publikation

Ziegler, J.; Stenzel, I.; Hause, B.; Maucher, H.; Hamberg, M.; Grimm, R.; Ganal, M.; Wasternack, C.; Molecular Cloning of Allene Oxide Cyclase J. Biol. Chem. 275, 19132-19138, (2000) DOI: 10.1074/jbc.M002133200

Allene oxide cyclase (EC 5.3.99.6) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5′-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (>99%) formation of the 9S,13Senantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.
Publikation

Kramell, R.; Miersch, O.; Schneider, G.; Wasternack, C.; Liquid chromatography of jasmonic acid amine conjugates Chromatographia 49, 42-46, (1999) DOI: 10.1007/BF02467185

Racemic jasmonic acid (3R,7R/3S,7S)-(±)-JA) was chemically conjugated with different biogenic amines originating from aliphatic and aromatic α-amino acids by decarboxylation. The resulting isomeric compounds were subjected to reversed-phase high-performance liquid chromatography (HPLC) and to HPLC on the chiral stationary phases Chiralpak AS and Nucleodex β-PM. Under reversed-phase conditions, all the homologous amine derivatives tested could be separated from each other except the JA-conjugates containing 2-phenyl-ethylamine and 3-methylbutylamine. On both chiral supports the (3R,7R)-(−)-JA conjugates eluted earlier than those of the enantiomeric counterpart (3S,7S)-(+)-JA. On Chiralpak AS all the isomers studied could be separated to baseline with a mobile phase containingn-hexane and 2-propanol. The calculated resolution factors were between 1.80 and 4.17. The pairs of isomers were also chromatographed on the cyclodextrin stationary phase Nucleodex β-PM with methanol-triethylammonium acetate buffer as mobile phase. Under these conditions resolution factors were between 0.74 and 1.29. The individual isomers were chiroptically characterized by measurement of their circular dichroism.
Publikation

Kramell, R.; Schneider, G.; Miersch, O.; Chiral separation of amide conjugates of jasmonic acid by liquid chromatography Chromatographia 45, 104-108, (1997) DOI: 10.1007/BF02505545

Synthetic amide conjugates of (−)-jasmonic acid and its (+)-enantiomer were resolved by means of chiral liquid chromatography. The diastereomeric pairs prepared by chemical reaction of (±)-jasmonic acid with a series of (S)- or (R)-amino acids and with some (S)-amino acid alcohols were completely separated on Chiralpak AS using a mixture of n-hexane/2-propanal as mobile phase. The retention data indicate that the (−)-jasmonic acid conjugates eluted faster than those of the (+)-enantiomer, independent on the configuration of the bound amino acid. Likewise, enantiomeric derivatives of (±)-jasmonic acid and non-chiral amino acids were completely separated on the chiral stationary phase and showed the same elution sequence. The resolution factors,Rs, were found to range between 1.13 and 6.64. The separated compounds were chiropatically analyzed by measurement of the circular dichroism.
Publikation

Hause, B.; Feussner, K.; Wasternack, C.; Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures Bot. Acta 110, 452-457, (1997) DOI: 10.1111/j.1438-8677.1997.tb00662.x

Diadenosine 5′,5′”‐P1,P4‐tetraphosphate (Ap4A) cleaving enzymes are assumed to regulate intracellular levels of Ap4A, a compound known to affect cell proliferation and stress responses. From plants an Ap4A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap4A hydrolase in 4‐day‐old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein‐5‐isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap4A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6‐diamidino‐2‐phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap4A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap4A in triggering DNA synthesis and cell proliferation.
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