Anaerobic initial reaction of n-alkanes in a denitrifying bacterium: evidence for (1-methylpentyl)succinate as initial product and for involvement of an organic radical in n-hexane metabolism.
A novel type of denitrifying bacterium (strain HxN1) with the capacity to oxidize n-alkanes anaerobically with nitrate as the electron acceptor to CO(2) formed (1-methylpentyl)succinate (MPS) during growth on n-hexane as the only organic substrate under strict exclusion of air. Identification of MPS by gas chromatography-mass spectrometry was based on comparison with a synthetic standard. MPS was not formed during anaerobic growth on n-hexanoate. Anaerobic growth with [1-(13)C]n-hexane or d(14)-n-hexane led to a 1-methylpentyl side chain in MPS with one (13)C atom or 13 deuterium atoms, respectively. This indicates that the 1-methylpentyl side chain originates directly from n-hexane. Electron paramagnetic resonance spectroscopy revealed the presence of an organic radical in n-hexane-grown cells but not in n-hexanoate-grown cells. Results point at a mechanistic similarity between the anaerobic initial reaction of n-hexane and that of toluene, even though n-hexane is much less reactive; the described initial reaction of toluene in anaerobic bacteria is an addition to fumarate via a radical mechanism yielding benzylsuccinate. We conclude that n-hexane is activated at its second carbon atom by a radical reaction and presumably added to fumarate as a cosubstrate, yielding MPS as the first stable product. When 2,3-d(2)-fumarate was added to cultures growing on unlabeled n-hexane, 3-d(1)-MPS rather than 2,3-d(2)-MPS was detected, indicating loss of one deuterium atom by an as yet unknown mechanism.
Rabus
R
R
Max-Planck-Institut für Marine Mikrobiologie, D-28359 Bremen, Germany.  rrabus@mpi-bremen.de
Wilkes
H
H
Behrends
A
A
Armstroff
A
A
Fischer
T
T
Pierik
A J
AJ
Widdel
F
F
D000473:Alkanes / Q000737:chemistry / Q000378:metabolism*; D000693:Anaerobiosis; D001421:Bacteria, Anaerobic / Q000254:growth & development* / Q000378:metabolism*; D003470:Culture Media; D004578:Electron Spin Resonance Spectroscopy; D008401:Gas Chromatography-Mass Spectrometry; D006586:Hexanes / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism*; D010084:Oxidation-Reduction; D013386:Succinates / Q000737:chemistry / Q000378:metabolism*
C422203:(1-methylpentyl)succinate; D000473:Alkanes; D003470:Culture Media; D006586:Hexanes; D009573:Nitrites; D013386:Succinates