title: The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway.
abstract: Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated beta-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.
authors.affiliation: Department of Environmental Microbiology, UFZ, Permoserstr. 15, 04318 Leipzig, Germany. thore.rohwerder@ufz.de
authors.forename: Thore
authors.initials: T
authors.lastname: Rohwerder
authors.forename: Uta
authors.initials: U
authors.lastname: Breuer
authors.forename: Dirk
authors.initials: D
authors.lastname: Benndorf
authors.forename: Ute
authors.initials: U
authors.lastname: Lechner
authors.forename: Roland H
authors.initials: RH
authors.lastname: Müller
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000378:metabolism*; D003470:Culture Media; D017931:DNA Primers; D005019:Ethyl Ethers / Q000378:metabolism*; D005742:Gasoline; D006885:Hydroxybutyrates / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009618:Nocardiaceae / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012242:Rhodobacter sphaeroides / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology; D014805:Vitamin B 12 / Q000494:pharmacology*; D020584:Xanthobacter / Q000378:metabolism
chemical_list: D003470:Culture Media; D017931:DNA Primers; D005019:Ethyl Ethers; D005742:Gasoline; D006885:Hydroxybutyrates; C008039:2-hydroxyisobutyric acid; D019751:Intramolecular Transferases; D014805:Vitamin B 12