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Characterization of a periplasmic quinoprotein from Sphingomonas wittichii that functions as aldehyde dehydrogenase

Applied Microbiology and Biotechnology volume 98, pages 2067–2079 (2014)Cite this article

Abstract

The α-proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit_4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5′ and 3′ ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit_4395 had a subunit mass of 39.3 kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1–C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with V max and apparent K M values of 3,970 U/mg protein and 12.3 mM, respectively. Pyrroloquinoline quinone was detected using UV–Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit_4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit_4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes.

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Acknowledgments

This project was supported by funds from Bundesministerium für Bildung und Forschung (BMBF, project no. 0315632A).

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Authors and Affiliations

  1. Institut für Mikrobiologie und Biotechnologie, 168 Meckenheimer Allee, 53115, Bonn, Germany

    Jessica Zeiser, Larissa Helen Mühlenbeck & Uwe Deppenmeier

  2. Biology Department, Missouri State University, 901 S. National Ave, Springfield, MO, 65897, USA

    Paul Schweiger

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  1. Jessica Zeiser
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  2. Larissa Helen Mühlenbeck
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  4. Uwe Deppenmeier
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Correspondence to Uwe Deppenmeier.

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Zeiser, J., Mühlenbeck, L.H., Schweiger, P. et al. Characterization of a periplasmic quinoprotein from Sphingomonas wittichii that functions as aldehyde dehydrogenase. Appl Microbiol Biotechnol 98, 2067–2079 (2014). https://doi.org/10.1007/s00253-013-5016-5

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  • DOI: https://doi.org/10.1007/s00253-013-5016-5

Keywords

  • Carboxylic acid
  • Oxidoreductase
  • Biotransformation
  • Detoxification
  • Stereospecific oxidation