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

Danny Brodkorb,Matthias Gottschall,Robert Marmulla,Frauke Lüddeke,Jens Harder

doi:10.1074/jbc.m109.084244

Danny Brodkorb, Matthias Gottschall + Show 3 more

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https://doi.org/10.1074/jbc.m109.084244

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Abstract

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V(max)) were 140 nanokatals mg(-1) for the linalool dehydratase and 410 nanokatals mg(-1) for the geraniol isomerase, with apparent K(m) values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) FR669447
Cultivation approaches established the enrichment of monoterpenemineralizing microorganisms (19) and the isolation of strains of Alcaligenes defragrans (20) and Thauera terpenica (21)
In this article we report on the detection of novel enzyme activities and the isolation and characterization of an anaerobic linalool dehydratase-isomerase, a bifunctional enzyme that catalyzes the reversible dehydration and isomerization of linalool (3,7-dimethyl-1,6-octadien-3-ol) (Fig. 1)

Summary

To whom correspondence should be addressed

The monoterpenes are divided into acyclic compounds, such as myrcene (7-methyl-3-methylene-1,6-octadiene) and ocimene, monocyclic monoterpenes, e.g. limonene and phellandrene, and bicyclic monoterpenes, e.g. pinene and sabinene. These unsaturated hydrocarbons are classified as highly volatile organic compounds. The mineralization of monoterpenes by aerobic microorganisms has been studied in detail with Pseudomonas species (10, 11). The anaerobic mineralization of monoterpenes to carbon dioxide is frequently present in denitrifying bacteria (18). 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). In this article we report on the detection of novel enzyme activities and the isolation and characterization of an anaerobic linalool dehydratase-isomerase, a bifunctional enzyme that catalyzes the reversible dehydration and isomerization of linalool (3,7-dimethyl-1,6-octadien-3-ol) (Fig. 1)

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DISCUSSION