Cloning,characterization, and mutational analysis of a highly active and stable <Emphasis Type="SmallCaps">l</Emphasis>-arabinitol 4-dehydrogenase from <Emphasis Type="Italic">Neurospora crassa</Emphasis> |
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Authors: | Ryan Sullivan " target="_blank">Huimin Zhao |
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Institution: | (1) Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA;(2) Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA;(3) Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA;(4) Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA |
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Abstract: | An NAD+-dependent l-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer and contained two Zn2+ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (SDHs). High enzymatic activity
was observed for substrates l-arabinitol, adonitol, and xylitol and no activity for d-mannitol, d-arabinitol, or d-sorbitol. The enzyme showed strong preference for NAD+ but also displayed a very low yet detectable activity with NADP+. Mutational analysis of residue F59, the single different substrate-binding residue between LADs and d-SDHs, failed to confer the enzyme the ability to accept d-sorbitol as a substrate, suggesting that the amino acids flanking the active site cleft may be responsible for the different
activity and affinity patterns between LADs and SDHs. This enzyme should be useful for in vivo and in vitro production of
xylitol and ethanol from l-arabinose.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Arabinose fermentation Xylitol production N crassa genome Alcohol dehydrogenase Homology modeling Ethanol production |
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