Geranic Acid Formation, an Initial Reaction of Anaerobic Monoterpene Metabolism in Denitrifying Alcaligenes defragrans

Abstract

Monoterpenes with an unsaturated hydrocarbon structure are mineralized anaerobically by the denitrifying ␤-proteobacterium Alcaligenes defragrans. Organic acids occurring in cells of A. defragrans and culture medium were characterized to identify potential products of the monoterpene activation reaction. Geranic acid (E,E-3,7-dimethyl-2,6-octadienoic acid) accumulated to 0.5 mM in cells grown on ␣-phellandrene under nitrate limitation. Cell suspensions of A. defragrans 65Phen synthesized geranic acid in the presence of ␤-myrcene, ␣-phellandrene, limonene, or ␣-pinene. Myrcene yielded the highest transformation rates. The alicyclic acid was consumed by cell suspensions during carbon limitation. Heat-labile substances present in cytosolic extracts catalyzed the formation of geranic acid from myrcene. These results indicated that a novel monoterpene degradation pathway must be present in A. defragrans.The mineralization of organic matter by microorganisms is often severely hampered by the chemical structure of the substrate. In the presence of oxygen, mono-and dioxygenases catalyze the oxidative functionalization of recalcitrant compounds, i.e., hydrocarbons. Microbial mineralization of such substances also occurs in nature in the absence of molecular oxygen, and in the last decade anaerobic bacteria have been isolated on substances such as alkylbenzenes (6, 18, 22), alkanes (1, 7, 25, 26), and alkenes (15). Thus far, the biochemistry of the initial activation reactions has been revealed only for toluene. Catalysis via radicals seems to be involved in the addition of toluene to fumarate by the enzyme benzylsuccinate synthase, a putative glycine radical enzyme (3,21). This novel enzyme raises the question of how the activation of alkanes and alkenes commences.Monoterpenes are ubiquitous alkenes in nature (Fig. 1). The physiological trait of anaerobic mineralization of monoterpenes to carbon dioxide has been demonstrated by the enrichment and isolation of denitrifying strains of Alcaligenes defragrans and Thauera terpenica (11,12,16). During growth of bicyclic monoterpenes, e.g., ␣-pinene, traces of monocyclic monoterpenes are detected, suggesting menthadienes as intermediates (19). A. defragrans cometabolically catalyzes the transformation of isolimonene to isoterpinolene. This 3,1-hydrogen-⌬ 1 -⌬ 3 -mutase reaction confirms the microbial activation of alkene bonds that are not polarized by adjacent functional groups. It also indicates the necessity for a sp 2 hybridization of the C-1 atom in order for microbial monoterpene oxidation to occur (19).It seems likely that the unsaturated monoterpenes may be transformed initially into an organic acid, because intermediates in catabolic pathways are usually organic acids, e.g., in the citrate cycle. If this acid cannot diffuse through membranes, the carbon source is fixed in the cell. To explore this hypothesis, we characterized the organic acids in cells and culture medium and studied the formation of geranic acid (3,7-dimethyl-2,6-trans,trans-octadienoic acid) in cells ...

Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

“…For biomass production, the strain was cultivated on 30 mM limonene and 100 mM nitrate (24). A 1-liter preculture was inoculated in a 10-liter vessel of carbonate-buffered mineral salt medium at pH 7.0.…”

“…Initial studies on potential metabolites of the degradation pathway identified isoterpinolene as metabolite that was apparently not further metabolized (23) and geranic acid as ionic intermediate present in nitrate-respiring cells that were grown on acyclic or cyclic monoterpenes, e.g. myrcene or limonene (24).…”

Geraniol and Geranial Dehydrogenases Induced in Anaerobic Monoterpene Degradation by Castellaniella defragrans

“…In C. defragrans, the linalool dehydratase-isomerase catalyzes the hydration of the acyclic ␤-myrcene to (S)-(ϩ)-linalool as well as the isomerization to geraniol (7,35). The formation of geranic acid was observed in vivo and in vitro (24), indicating the presence of dehydrogenases catalyzing the oxidation of the allyl alcohol geraniol to geranic acid, most probably via geranial. So far, the only geraniol dehydrogenases (GeDHs) characterized on the molecular level come from sweet basil, Ocimum basilicum (25), and the astigmatid mite Carpoglyphus lactis (37).…”

Biochemical characterization of AtuD from Pseudomonas aeruginosa, the first member of a new subgroup of acyl-CoA dehydrogenases with specificity for citronellyl-CoA

“…Some information exists on conversion of terpenes under anaerobic conditions (Harder & Probian, 1995;Hylemon & Harder, 1998). Acyclic terpenes such as geranylate have been described as metabolites of cyclic terpene-utilizing anaerobes (Heyen & Harder, 2000). Aerobically, citronellol can be used as a single source of carbon and energy only by Pseudomonas citronellolis (Seubert, 1960;Seubert & Fass, 1964a, b) and a few related species such as Pseudomonas aeruginosa, Pseudomonas mendocina (Cantwell et al, 1978) and Pseudomonas delhiensis (Prakash et al, 2007).…”