Wolfgang Brandt

A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding to the F-box protein TIR1 and promotes the degradation of the Aux/IAA transcriptional repressors. Here, we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity appears to be largely determined by the Aux/IAA. As there are 6 TIR1/AFBs and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin sensing properties. We also demonstrate that the AFB5-Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response.

Virtual screening for plant PARP inhibitors – what can be learned from human PARP inhibitors?

The functions of Poly(ADP-ribose) polymerase enzymes (PARPs) in general are best studied based on human PARP-1 (HsPARP-1). HsPARP-1 is well investigated because pharmacological modulation of its activity modulates DNA-binding of antitumor drugs [1]. In contrast to human PARP enzymes, the exact role of PARPs in plants remains to be elucidated. Different stresses activate plant PARP enzymes to mediate DNA repair and (programmed) cell death whereas the addition of PARP inhibitors decreases the degree of cell death [2]. Therefore, the development of plant PARP inhibitors might be a way to increase the tolerance against abiotic stress. Initial to searches in commercial databases for potential plant PARP inhibitors, a virtual screening route had to be established for human PARP-1 inhibitors. Simultaneously, every step in that procedure was applied on a plant PARP enzyme to investigate the differences of both active sites. All differences have been evaluated statistically, e.g. using receiver-operator characteristics (ROC) and power analyses. At the end of that parallel screening route, a docking threshold for Arabidopsis thaliana L. PARP-1 (AtPARP-1) could be derived by knowledge transfer from the corresponding human receptor and its inhibitors. Figure 1 Key steps in virtual screening routes for human and Arabidopsis thaliana L. PARP-1. The results have been used to successfully apply the screening process for AtPARP-1 on a commercial database. Knowing the differences of the human and plant screening routes, predictions of the applicability of that multi-step process on a commercial database have been explored. Finally, the developed virtual screening route has been applied to screen a commercial database for AtPARP-1 inhibitors. From 20 compounds tested so far in vitro, 13 show inhibitory effects.

What can a chemist learn from nature's macrocycles?--a brief, conceptual view.

Macrocyclic natural products often display remarkable biological activities, and many of these compounds (or their derivatives) are used as drugs. The chemical diversity of these compounds is immense and may provide inspiration for innovative drug design. Therefore, a database of naturally occurring macrocycles was analyzed for ring size, molecular weight distribution, and the frequency of some common substructural motifs. The underlying principles of the chemical diversity are reviewed in terms of biosynthetic origin and nature’s strategies for diversity and complexity generation in relation to the structural diversity and similarities found in the macrocycle database. Finally, it is suggested that synthetic chemists should use not only nature’s molecules, but also nature’s strategies as a source of inspiration. To illustrate this, the biosynthesis of macrocycles by non-ribosomal peptide synthetases and terpene and polyketide cyclases, as well as recent advances of these strategies in an integrated synthesis/biotechnology approach are briefly reviewed.

Evolution of morphine biosynthesis in opium poppy.

Benzylisoquinoline alkaloids (BIAs) are a group of nitrogen-containing plant secondary metabolites comprised of an estimated 2500 identified structures. In BIA metabolism, (S)-reticuline is a key branch-point intermediate that can be directed into several alkaloid subtypes with different structural skeleton configurations. The morphinan alkaloids are one subclass of BIAs produced in only a few plant species, most notably and abundantly in the opium poppy (Papaver somniferum). Comparative transcriptome analysis of opium poppy and several other Papaver species that do not accumulate morphinan alkaloids showed that known genes encoding BIA biosynthetic enzymes are expressed at higher levels in P. somniferum. Three unknown cDNAs that are co-ordinately expressed with several BIA biosynthetic genes were identified as enzymes in the pathway. One of these enzymes, salutaridine reductase (SalR), which is specific for the production of morphinan alkaloids, was isolated and heterologously overexpressed in its active form not only from P. somniferum, but also from Papaver species that do not produce morphinan alkaloids. SalR is a member of a class of short chain dehydrogenase/reductases (SDRs) that are active as monomers and possess an extended amino acid sequence compared with classical SDRs. Homology modelling and substrate docking revealed the substrate binding site for SalR. The amino acids residues conferring salutaridine binding were compared to several members of the SDR family from different plant species, which non-specifically reduce (-)-menthone to (+)-neomenthol. Previously, it was shown that some of these proteins are involved in plant defence. The recruitment of specific monomeric SDRs from monomeric SDRs involved in plant defence is discussed.

UBIAD1 Mutation Alters a Mitochondrial Prenyltransferase to Cause Schnyder Corneal Dystrophy

Background Mutations in a novel gene, UBIAD1, were recently found to cause the autosomal dominant eye disease Schnyder corneal dystrophy (SCD). SCD is characterized by an abnormal deposition of cholesterol and phospholipids in the cornea resulting in progressive corneal opacification and visual loss. We characterized lesions in the UBIAD1 gene in new SCD families and examined protein homology, localization, and structure. Methodology/Principal Findings We characterized five novel mutations in the UBIAD1 gene in ten SCD families, including a first SCD family of Native American ethnicity. Examination of protein homology revealed that SCD altered amino acids which were highly conserved across species. Cell lines were established from patients including keratocytes obtained after corneal transplant surgery and lymphoblastoid cell lines from Epstein-Barr virus immortalized peripheral blood mononuclear cells. These were used to determine the subcellular localization of mutant and wild type protein, and to examine cholesterol metabolite ratios. Immunohistochemistry using antibodies specific for UBIAD1 protein in keratocytes revealed that both wild type and N102S protein were localized sub-cellularly to mitochondria. Analysis of cholesterol metabolites in patient cell line extracts showed no significant alteration in the presence of mutant protein indicating a potentially novel function of the UBIAD1 protein in cholesterol biochemistry. Molecular modeling was used to develop a model of human UBIAD1 protein in a membrane and revealed potentially critical roles for amino acids mutated in SCD. Potential primary and secondary substrate binding sites were identified and docking simulations indicated likely substrates including prenyl and phenolic molecules. Conclusions/Significance Accumulating evidence from the SCD familial mutation spectrum, protein homology across species, and molecular modeling suggest that protein function is likely down-regulated by SCD mutations. Mitochondrial UBIAD1 protein appears to have a highly conserved function that, at least in humans, is involved in cholesterol metabolism in a novel manner.

The Functional Role of Selenocysteine (Sec) in the Catalysis Mechanism of Large Thioredoxin Reductases: Proposition of a Swapping Catalytic Triad Including a Sec‐His‐Glu State

Thioredoxin reductases catalyse the reduction of thioredoxin disulfide and some other oxidised cell constituents. They are homodimeric proteins containing one FAD and accepting one NADPH per subunit as essential cofactors. Some of these reductases contain a selenocysteine at the C terminus. Based on the X‐ray structure of rat thioredoxin reductase, homology models of human thioredoxin reductase were created and subsequently docked to thioredoxin to model the active complex. The formation of a new type of a catalytic triad between selenocysteine, histidine and a glutamate could be detected in the protein structure. By means of DFT (B3LYP, lacv3p**) calculations, we could show that the formation of such a triad is essential to support the proton transfer from selenol to a histidine to stabilise a selenolate anion, which is able to interact with the disulfide of thioredoxin and catalyses the reductive disulfide opening. Whereas a simple proton transfer from selenocysteine to histidine is thermodynamically disfavoured by some 18 kcal mol−1, it becomes favoured when the carboxylic acid group of a glutamate stabilises the formed imidazole cation. An identical process with a cysteine instead of selenocysteine will require 4 kcal mol−1 more energy, which corresponds to a calculated equilibrium shift of ∼1000:1 or a 103 rate acceleration: a value close to the experimental one of about 102 times. These results give new insights into the catalytic mechanism of thioredoxin reductase and, for the first time, explain the advantage of the incorporation of a selenocysteine instead of a cysteine residue in a protein.

Functional characterization of a novel benzylisoquinoline O‐methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L)

The benzylisoquinoline alkaloids are a highly diverse group of about 2500 compounds which accumulate in a species-specific manner. Despite the numerous compounds which could be identified, the biosynthetic pathways and the participating enzymes or cDNAs could be characterized only for a few selected members, whereas the biosynthesis of the majority of the compounds is still largely unknown. In an attempt to characterize additional biosynthetic steps at the molecular level, integration of alkaloid and transcript profiling across Papaver species was performed. This analysis showed high expression of an expressed sequence tag (EST) of unknown function only in Papaver somniferum varieties. After full-length cloning of the open reading frame and sequence analysis, this EST could be classified as a member of the class II type O-methyltransferase protein family. It was related to O-methyltransferases from benzylisoquinoline biosynthesis, and the amino acid sequence showed 68% identical residues to norcoclaurine 6-O-methyltransferase. However, rather than methylating norcoclaurine, the recombinant protein methylated norreticuline at position seven with a K(m) of 44 mum using S-adenosyl-l-methionine as a cofactor. Of all substrates tested, only norreticuline was converted. Even minor changes in the benzylisoquinoline backbone were not tolerated by the enzyme. Accordingly, the enzyme was named norreticuline 7-O-methyltransferase (N7OMT). This enzyme represents a novel O-methyltransferase in benzylisoquinoline metabolism. Expression analysis showed slightly increased expression of N7OMT in P. somniferum varieties containing papaverine, suggesting its involvement in the partially unknown biosynthesis of this pharmaceutically important compound.

A Structural Model of the Membrane‐Bound Aromatic Prenyltransferase UbiA from E. coli

The membrane‐bound enzyme 4‐hydroxybenzoic acid oligoprenyltransferase (ubiA) from E. coli is crucial for the production of ubiquinone, the essential electron carrier in prokaryotic and eukaryotic organisms. On the basis of previous modeling analyses, amino acids identified as important in two putative active sites (1 and 2) were selectively mutated. All mutants but one lost their ability to form geranylated hydroxybenzoate, irrespective of their being from active site 1 or 2. This suggests either that the two active sites are interrelated or that they are in fact only one site. With the aid of the experimental results and a new structure‐based classification of prenylating enzymes, a relevant 3D model could be developed by threading. The new model explains the substrate specificities and is in complete agreement with the results of site‐directed mutagenesis. The high similarity of the active fold of UbiA‐transferase to that of 5‐epi‐aristolochene synthase (Nicotiana tabacum), despite a low homology, allows a hypothesis on a convergent evolution of these enzymes to be formed.

Benzoxazinoid biosynthesis in dicot plants

Benzoxazinoids are common defence compounds of the grasses and are sporadically found in single species of two unrelated orders of the dicots. In the three dicotyledonous species Aphelandra squarrosa, Consolida orientalis and Lamium galeobdolon the main benzoxazinoid aglucon is 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA). While benzoxazinoids in Aphelandra squarrosa are restricted to the root, in Consolida orientalis and Lamium galeobdolon DIBOA is found in all above ground organs of the adult plant in concentrations as high as in the seedling of maize. The initial biosynthetic steps in dicots and monocots seem to be identical. Indole is most probably the first specific intermediate that is oxygenated to indolin-2-one by a cytochrome P450 enzyme. C. orientalis has an active indole-3-glycerolphosphate lyase for indole formation that evolved independently from its orthologous function in maize. The properties and evolution of plant indole-3-glycerolphosphate lyases are discussed.

Inhibitors for Human Glutaminyl Cyclase by Structure Based Design and Bioisosteric Replacement

The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimers disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.