title: Linalool isomerase, a membrane-anchored enzyme in the anaerobic monoterpene degradation in Thauera linaloolentis 47Lol.
abstract: BACKGROUND
  Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Δ(1)-Δ(2)-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool.
  
  
  RESULTS
  The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42 ± 0.28 nkat mg * protein(-1) and a kM value of 455 ± 124 μM were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 °C.
  
  
  CONCLUSIONS
  The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers.
authors.affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology, Celsiusstr. 1, D-28359, Bremen, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Marmulla
authors.affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology, Celsiusstr. 1, D-28359, Bremen, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Šafarić
authors.affiliation: Institute for Pharmacy, Department of Pharmaceutical Biotechnology, University of Greifswald, Felix-Hausdorff-Str. 3, D-17487, Greifswald, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Markert
authors.affiliation: Institute for Pharmacy, Department of Pharmaceutical Biotechnology, University of Greifswald, Felix-Hausdorff-Str. 3, D-17487, Greifswald, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schweder
authors.affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology, Celsiusstr. 1, D-28359, Bremen, Germany. jharder@mpi-bremen.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Harder
mesh_terms: D000080462:Acyclic Monoterpenes; D002473:Cell Wall / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006836:Hydro-Lyases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D039821:Monoterpenes / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013104:Spheroplasts / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism; D020628:Thauera / Q000737:chemistry / Q000201:enzymology*
chemical_list: D000080462:Acyclic Monoterpenes; D039821:Monoterpenes; D011994:Recombinant Proteins; D013729:Terpenes; C018584:linalool; D006836:Hydro-Lyases; D007535:Isomerases; C007836:geraniol
keywords: Allyl alcohol; Geraniol; Isomerase; Linalool; Monoterpene; Thauera