Biotechnologie

4-Hydroxybenzoate oligoprenyl transferase from E. coli (ubiA-prenyl transferase) is a crucial enzyme for ubiquinone biosynthesis. It catalyzes the formation of 3-oligoprenyl-4-hydroxybenzoates like geranyl hydroxybenzoate (GHB, 23) from geranyl pyrophosphate (GPP, 22). Several analogues and mimics of geranyl pyrophosphate have been prepared for an examination of their ability to inhibit the enzyme. 7,11-Dimethyl-3-oxododeca-6,10-dienoic acid (2), 3-hydroxy7,11- dimethyldodeca-6,10-dienoic acid (3), 2-hydroxy-4,8-dimethyl-3,7-nonadienylphosphonic acid (4), and tripotassium [[(4E)-5,9-dimethyldeca-4,8-dienyl]phosphinato](difluoro)methylphosphonate (5) were synthesized from geraniol. .-2,.-1-Dihydroxylated farnesyl diphosphate 6 was prepared from trans,trans-farnesol. All compounds were tested for enzyme inhibition in a competitive assay with natural substrate. The effect of these compounds on ubiA-prenyltransferase activity varied substantially, ranging from almost full inhibition to, surprisingly, enhanced enzymatic activity at low concentrations by some compounds. A special, EDTAmodifyable magnesium effect is discussed as potential reason.

The aminocoumarin antibiotics clorobiocin, novobiocin, and coumermycin A1 are inhibitors of bacterial gyrase. Their chemical structures contain amide bonds, formed between an aminocoumarin ring and an aromatic acyl component, which is 3-dimethylallyl-4-hydroxybenzoate in the case of novobiocin and clorobiocin. These amide bonds are formed under catalysis of the gene products of cloL, novL, and couL, respectively. We first examined the substrate specificity of the purified amide synthetases CloL, NovL, and CouL for the various analogs of the prenylated benzoate moiety. We then generated new aminocoumarin antibiotics by feeding synthetic analogs of the 3-dimethylallyl-4-hydroxybenzoate moiety to a mutant strain defective in the biosynthesis of the prenylated benzoate moiety. This resulted in the formation of 32 new aminocoumarin compounds. The structures of these compounds were elucidated using FAB-MS and 1H-NMR spectroscopy.

Experimental and theoretical investigations concerning the second‐to‐last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4‐oxo‐DMAPP ( 12 ) and 4‐hydroxy‐DMAPP ( 11 ) were synthesized in deuterium‐ or tritium‐labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP ( 7 ), 11 , and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three‐dimensional model of the fragment L271–A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe 4 S 4 cluster, and MEcPP ( 7 ) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP ( 7 ), hydroxyl abstraction, and formation of HMBPP ( 8 ). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe 4 S 4 cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP ( 8 ) to IPP ( 9 ) and DMAPP ( 10 ) in the last step.

Tocopherols and tocotrienols are phytochemicals generally known as vitamin E. Structurally they are very similar sharing an identical aromatic moiety (chromanol head) and an isoprenoid side chain that only differs in the degree of saturation. Tocopherols are widely distributed throughout the plant kingdom, found in practically all plant tissues and located in the inner envelope membrane of plastids. In contrast to these well characterized tocopherols little is known on the presence, location and synthesis of tocotrienols. Studies so far have been hampered by the lack of a suitable model tissue in which high tocotrienol synthesis rates could be demonstrated. Here it is shown irrevocably that tocotrienols are present in seeds of mature grapes at a relatively high concentration of (33,2 +/- 6,0) μg tocotrienols / g dry weight. Showing a distribution pattern between the - and - isomers of respectively 40 and 60 %. These data are in strong contrast with the general idea that tocotrienols are only present in monocotyledonous plants. In leaves or flowers of the grape plants on the other hand, no measurable tocotrienol concentrations were detected. Tocopherols were present in all tested tissues with highest concentrations in green leaves (30 μg tocopherols / g dry weight) and lowest in flesh of grapes (5 μg tocotrienols / g dry weight). Oil extracted from the grapes was 25-fold enriched in tocotrienols. To study the biosynthesis of tocotrienols a new procedure was developed mainly differing from original methods described in Methods of enzymology by threlfall et al. who worked on complete plants, and by Soll working on chloroplasts extracted from green tissues, none of the plant tissues Threlfall and Soll discribe produce tocotrienols. With the use of radioactive labeled precursors de novo synthesis of tocotrienols in dissected endosperm of developing grape seeds (45 DAF) was demonstrated. The use of endosperm of developing grapes as a model tissue to study tocotrienol location, biosynthesis and function is discussed.

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The missing link in the new deoxyxylulose phosphate metabolic pathway leading to the biosynthesis of plant terpenoids has been identified. The intermediate between the cyclic diphosphate 1 and the basic isoprenoid building blocks dimethylallyl diphosphate and isopentenyl diphosphate has been shown for the first time to be (E )‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate (2 ) by incorporation of tritium‐labeled 2 into phytoene.

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Die enzymatische Friedel‐Crafts‐Alkylierung zur Synthese bis zu fünffach kernsubstituierter 3‐Polyprenylbenzoesäurederivate gelingt regiospezifisch mit einer Polyprenyl‐Transferase, die ein erstaunlich breites Substratspektrum aufweist [Gl. (a); PP = Pyrophosphat]. Obwohl es sich um ein Membranenzym handelt, sind wegen der einfachen Gewinnung und Anreicherung sowie der guten Lagerstabilität die Voraussetzungen für den Einsatz in der organischen Synthese ausgezeichnet.

The enzymatic Friedel–Crafts alkylation for the synthesis of up to fivefold ring‐substituted 3‐polyprenylbenzoic acid derivatives can be accomplished regiospecifically with a polyprenyl‐transferase, which exhibits a surprisingly broad substrate spectrum ([Eq. (a)]; OPP = pyrophosphate). Although a membrane enzyme, its simple production and enrichment, and good shelf life provide an excellent basis for general use in organic synthesis.