Die genetische und biochemische Charakterisierung von Enzymen des anaeroben Monoterpen-Abbaus in Castellaniella defragrans

Castellaniella defragrans is a betaproteobacterium metabolizing monoterpenes anaerobically under denitrifying conditions. Metabolite studies as well as a differential proteomic approach led to the identification of a linalool dehydratase-isomerase (ldi/LDI), a geraniol dehydrogenase (gedh/GeDH) and a candidate gene for a geranial dehydrogenase (gadh/GaDH). The LDI and GeDH were subjected to an initial biochemical characterization. In order to gain insight into the in vivo function of these genes a genetic system for C. defragrans was developed based on a suicide vector. After confirmation of the correct genetic background the physiological characaterization of the obtained deletionmutants verified the postulated degradation pathway for β-myrcene. Besides, the results disclosed another monoterpene degradation pathway as well as novel enzyme activities. In addition to the genetic and physiological characterization the enzymes were heterologously expressed in E. coli and biochemically studied. The bifunctionally LDI catalyzes enantiospecifically the hydration of β-myrcene to (S)-( )-linalool with an enantiomeric excess of > 95 % that is further isomerised to geraniol. Chemical modification of cysteine, histidine, aspartic and glutamic acid residues inhibited the enzyme activity. By the deletion of the ldi gene in C. defragrans a phenotype was caused that lost the ability of degrading the acyclic β-myrcene, but cyclic monoterpene degradation was not effected. A novel enzyme activity acting on (R,S)-(±)-linalool was observed, too. The heterologously expressed GeDH was purified to homogeneity and native PAGE in combination with an activity staining revealed the native conformation as dimer. With regards to their kinetic properties recombinant and wild type GeDH correlated well with a high affinity for geraniol. Due to the deletion of the gedh mutant strains revealed an impaired growth on monoterpenes suggesting the presence of further alcohol dehydrogenases acting on geraniol. The candidate gene for a geranial dehydrogenase, which was found to be induced by growth on monoterpenes, interceded specifically the oxidation of geranial. The cis-isomer was not converted. The formation of NAD was in a stoichiometric ratio. To our knowledge, both GeDH and GaDH are the first monoterpenoid specific dehydrogenases originating from a bacterium.