Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans.

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprenes, and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here, we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a DTT:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently, the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism (ctm) in C. defragrans and are annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB, and CtmAB in Escherichia coli demonstrated that limonene dehydrogenase activity required both subunits, each carrying a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest placing in Enzyme Nomenclature as new entry EC 1.17.99.8.