Bactobilin: blue bile pigment isolated from Clostridium tetanomorphum.

Abstract

A blue bile pigment, possessing four acetic and four propionic acid side chains has been isolated from extracts of the anaerobic microorganism Clostridium tetanomorphum and in smaller amounts from Propionibacterium shermanii. The compound could be prepared in larger amounts by incubation of C. tetanomorphum enzyme extracts with added 8-aminolevulinic acid. The ultraviolet-visible, infrared, and proton magnetic resonance spectra of the pigment indicate a chromophore of the biliverdin type. Field-desorption mass spectrometry of the purified methyl ester showed a strong molecular ion at m/e = 962. This corresponds to the molecular weight expected for the octamethyl ester of a bilatriene type of bile pigment structurally derived from uroporphyrin 1m or I. Of the five possible structures, two could be eliminated by proton magnetic resonance spectroscopy. The name bactobilin is proposed for this previously unreported bile pigment.The open-chain tetrapyrrole compounds known as bile pigments are widely distributed. They are found free or bound to protein in mammals, birds, amphibians, reptiles, fish, molluscs, and insects and in algae and higher plants (for reviews, see refs. 1-9). As side chains or 1&carbon ring substituents, all bile pigments described thus far have four methyl groups, two propionic acid groups, and two vinyl groups, one or both of which can be isomerized to ethylidene (4, 10, 11) or reduced to ethyl (6,10,11). These substituents are arranged in the sequence found in protoporphyrin IX; in fact, all these bile pigments are formed from protoheme (8,(12)(13)(14), whose oxidative breakdown to biliverdin is catalyzed by the enzyme heme oxygenase (15-17). Bile pigments thus far have not been detected in prokaryotes. The present paper reports the isolation and in vitro formation of a bile pigment from the bacterium Clostridium tetanomorphum. This anaerobic organism makes uroporphyrinogen, the precursor of vitamin B-12, but not heme, protoporphyrinogen, or coproporphyrinogen. The isolated bile pigment is of interest not only because of its detection in a prokaryote, but also because its 13-carbon ring substituents, four acetic acid and four propionic acid groups, correspond to those of a uroporphyrin and not of protoporphyrin. MATERIALS AND METHODSSupplies. -Aminolevulinic acid was obtained from Sigma;biliverdin IXa dimethyl ester and the fully esterified methyl esters of uroporphyrin III, coproporphyrin III, protoporphyrin IX, and heptacarboxylporphyrin I, were from Porphyrin Products (Logan, UT). The n-hexane used for flash chromatography was "95 + %" (Aldrich), whereas the n-hexane for thin-layer chromatography was 99 mol % pure (Fisher). Other chemicals used were reagent grade or better. Silica gel for flash chromatography and a flash chromatography column, 3.4-cm diameter, were obtained from J. T. Baker. Whatman high-performance silica thin-layer chromatography plates with a preadsorbent spotting area (type LHP-K, 10 x 20 cm) and E. Merck precoated cellulose thin-layer chromatography plates were...

Characterization of a Heme-Dependent Catalase from Methanobrevibacter arboriphilus

“…Strain AZ has been isolated from digested sludge of an anaerobic sewage treatment plant near Zürich, Switzerland (38), and the type strain DH1 has been isolated from wet-wood cores of trees growing near Madison, Wis. (39). Hemin is predicted to be available at relatively high concentrations in both anaerobic habitats, since in anaerobic environments heme released upon degradation of heme-containing proteins is only very slowly metabolized: known pathways require molecular oxygen to initiate heme degradation (8). It is therefore very likely that both M. arboriphilus strains also synthesize active catalase in their natural environments despite the fact that they cannot synthesize heme.…”

Anaerobic breakdown of uroporpophysrins I and II to bile pigments by extracts of Clostridium tetanomorphum

“…C. tetanomorphum (ATCC 19407) was grown overnight without agitation at 37°C in stoppered 1 1 bottles on a medium described earlier [1,8].…”

“…A previous report [1] demonstrated the formation of an unusual blue bile pigment, bactobilin, when an enzyme preparation from Clostridium tetanomorphum was incubated with the tetrapyrrole precursor 8-aminolevulinate. This substance was the first bile pigment from a bacterial source and, in contrast to all eukaryotic bile pigments, it contained not two but eight carboxyl groups.…”

“…it was a urobiliverdin [5]. A comparison of the 1H NMR spectra of bactobilin octamethyl ester [1] with the 1H NMR spectra of the octamethyl esters of the five synthetic urobiliverdins derivable from uroporphyrins I and III unexpectedly established the side chains of bactobilin to have the repetitive alternating acetic acid, propionic acid sequence of uroporphyrin I [6]. The present work was undertaken to determine whether bacterial bile pigments related to the more prevalent uroporphyrin III can be formed as well.…”

“…C. tetanomorphum makes uroporphyrinogen III as corrinoid precursor [4], but it does not make heme, protoporphyrin IX, or uroporphyrins I and III. Optical, mass-spectral and 1H NMR evidence established bactobilin to be a bilatriene with the carboxylic side chains characteristic of a uroporphyrin [1], i.e. it was a urobiliverdin [5].…”