A member of the amidohydrolase superfamily, (BmulJ_04915 from Burkholderia multivorans), of unknown function was determined to hydrolyze a small set of sugar lactones: L-fucono-1,4-lactone, D-arabino-1,4-lactone, L-xylono-1,4-lactone, D-lyxono-1,4-lactone and L-galactono-1,4-lactone.  The highest activity was shown for L-fucono-1,4-lactone with a kcat of 1.1 s-1and a kcat/Km of 1.8 x 104 M-1 s-1.  The enzymatic product of an adjacent L-fucose dehydrogenase (BmulJ_04919) was shown to be L-fucono-1,5-lactone, via NMR spectroscopy.  L-fucono-1,5-lactone is unstable and rapidly converts non-enzymatically to L-fucono-1,4-lactone.  Due to the chemical instability of L-fucono-1,5-lactone, 4-deoxy-L-fucono-1,5-lactone was enzymatically synthesized  from 4-deoxy-L-fucose using L-fucose dehydrogenase. BmulJ_04915 hydrolyzed 4-deoxy-L-fucono-1,5-lactone with a kcat of 370 s-1 and a kcat/Km of 8.3 x 106 M-1s-1.  Inductively coupled plasma-mass spectrometery analysis of purified BmulJ_04915 showed 0.7 molar equivalents of Zn 2+ associated with the protein. However, when the metal was removed using chelating agents there was no significant change in catalytic activity.  The addition of divalent metals (Mn 2+, Zn 2+, Co2+, Cu2+ or Ni2+) also provided no significant change in the catalytic activity of this enzyme.  BmulJ_04915 is the second member of cog3618 that does not require a divalent metal for catalysis, alongside 2-pyrone-4,6-dicarboxylate lactonase (LigI).  To the best of our knowledge, BmulJ_04915 is the first enzyme shown to catalyze the hydrolysis of L-fucono-1,4-lactone or L-fucono-1,5-lactone.

Discovery of an L-fucono-1,5-lactonase within cog3618 from the amidohydrolase superfamily

244th National Meeting (2012)

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