Jürgen Wörth

Aerobic benzoyl-CoA catabolic pathway in Azoarcus evansii: studies on the non-oxygenolytic ring cleavage enzyme

A novel aerobic benzoate pathway has recently been discovered in various bacteria in which benzoate is first converted to benzoyl‐CoA. The further downstream steps are associated with the gene products of the benzoate oxidation gene cluster (box) on the Azoarcus evansii chromosome. Benzoyl‐CoA is oxidized to 2,3‐dihydro‐2,3‐dihydroxybenzoyl‐CoA (benzoyl‐CoA dihydrodiol) by benzoyl‐CoA oxygenase/reductase BoxBA in the presence of molecular oxygen. This study identified the next, ring cleaving step catalysed by BoxC. The boxC gene was expressed in a recombinant Escherichia coli strain as a fusion protein with maltose binding protein (BoxCmal) and the wild type as well as the recombinant proteins were purified and studied. BoxC catalyses the reaction 2,3‐dihydro‐2,3‐dihydroxybenzoyl‐CoA + H2O → 3,4‐dehydroadipyl‐CoA semialdehyde + HCOOH. This is supported by the following results. Assays containing [ring‐13C6]benzoyl‐CoA, benzoyl‐CoA oxygenase/reductase, BoxCmal protein, NADPH and semicarbazide were analysed directly by NMR spectroscopy and mass spectrometry. The products were identified as the semicarbazone of [2,3,4,5,6‐13C5]3,4‐dehydroadipyl‐CoA semialdehyde; the missing one‐carbon unit being formate. The same reaction mixture without semicarbazide yielded a mixture of the hydrate of [2,3,4,5,6‐13C5]3,4‐dehydroadipyl‐CoA semialdehyde and [2,3,4,5,6‐13C5]4,5‐dehydroadipyl‐CoA semialdehyde. BoxC, a 122 kDa homodimeric enzyme (61 kDa subunits), is termed benzoyl‐CoA‐dihydrodiol lyase. It contains domains characteristic for enoyl‐CoA hydratases/isomerases, besides a large central domain with no significant similarity to sequences in the database. The purified protein did not require divalent metals, molecular oxygen or any cosubstrates or coenzymes for activity. The complex reaction is part of a widely distributed new principle of aerobic aromatic metabolism in which all intermediates are coenzyme A thioesters and the actual ring‐cleavage reaction does not require molecular oxygen.

Aerobic benzoyl-coenzyme A (CoA) catabolic pathway in Azoarcus evansii: conversion of ring cleavage product by 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase.

Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde was studied in the beta-proteobacterium Azoarcus evansii. Cell extracts contained a benzoate-induced, NADP(+)-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in Escherichia coli, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.

From Pagodanes to Dodecahedranes - Search for a Serviceable Access to the Parent (C20H20) Hydrocarbon**

Abstract By taking advantage of the novel SN2 pagodane → dodecahedrane routes a preparatively potent access to the parent pentagonal dodecahedrane (2) was explored. The one-pot catalytic procedure (Pd/C/H2) starting from an eightfold functionalized secopagodane (14, C20H12Br6 (CO2 CH3)2) excells in shortness but, rather erratic (33–53%), falls out of the competition. The longer route via 1,6-dicarboxyl/dibromo dodecahedranes (35, 22) turned out as first choice with a total yield of 74–76% based on the common pagodane precursor (13).

Mitomycin und ein neues Phenoxazon-Pigment aus Streptomyces michiganensis

Streptomyces michiganensis strain Tü 1074, was isolated from a Tunesian soil sample and produces in liquid medium an antibiotic active pigment complex. Besides mitomycin A the separation of this complex yielded a nonactive phenoxazone, which hitherto has not been described in the literature. In contrary to all known phenoxazones from micro-organisms the new compound lacks a 2-amino-function. The production of this phenoxazone could be enhanced by optimizing the condtions of fermentation.Streptomyces michiganensis strain Tü 1074, was isolated from a Tunesian soil sample and produces in liquid medium an antibiotic active pigment complex. Besides mitomycin A the separation of this complex yielded a nonactive phenoxazone, which hitherto has not been described in the literature. In contrary to all known phenoxazones from microorganisms the new compound lacks a 2-amino-function. The production of this phenoxazone could be enhanced by optimizing the conditions of fermentation.ZusammenfassungDer aus einer tunesischen Bodenprobe isolierte Streptomyceten-Stamm Tü 1074 bildet in flüssigem Nährmedium einen antibiotisch wirksamen Pigmentkomplex. Bei der Auftrennung dieses Pigmentkomplexes fiel neben Mitomycin A ein antibiotisch unwirksames Phenoxazon an, das bisher noch nicht beschrieben wurde und im Gegensatz zu den bekannten Phenoxazonen aus Mikroorganismen keine 2-Amino-Funktion besitzt. Die Produktion des neuen Phenoxazons konnte durch Änderung der Fermentationsbedingungen optimiert werden.

Pentagonal dodecahedranes. Novel substitution patterns— MS fragmentation and unsaturation

For two- to six-fold functionalised dodecahedranes (7–10) the chances for selective variation of their substitution have been explored, as part of a program directed at homododecahedranes and at highly unsaturated dodecahedranes, ultimately C20 fullerene. With 1,6-dimethyl ester 7 several side chain transformations next to the very bulky dodecahedral cage were effected (1,6-bismethylene derivatives 25–31). In changing environments, Barton-type halogenative/hydrogenative decarboxylations (15–17, 38, 49, 59, 60, 77) as well as various nucleophilic substitutions (18, 20, 23, 39, 61–64) were achieved, mostly with good to high efficiency and retention of the substitution patterns. For the cage olefins 8/9 and the diepoxide 10, front-side cis-1,2-addition faced only slight competition in the reactions with HBr and CF3CO2H, but was only a minor pathway in the reaction with Br2. In the latter case, by a sequence of Br+ addition/HBr elimination steps, up to nine vicinally placed bromine substituents were implanted upon the C20 skeleton. The fate of variously functionalised dodecahedranes upon electron impact was studied—the competition between external (C–X/Y) and internal (C–C) bond cleavage was found to be typically dependent on the nature and relative orientation of the functionalities (X,Y) involved. There is good evidence that ions between m/z 242 (C20H2) and 256 (C20H16) resulting from the elimination of the respective Br, CO2CH3, CO2NH2, OCOCF3 substituents represent unsaturated dodecahedranes with up to nine CC double bonds.