Applied Microbiology and Biotechnology

, Volume 101, Issue 4, pp 1499–1507 | Cite as

Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases

  • Anders M. Knight
  • Alberto Nobili
  • Tom van den Bergh
  • Maika Genz
  • Henk-Jan Joosten
  • Dirk Albrecht
  • Katharina Riedel
  • Ioannis V. Pavlidis
  • Uwe T. Bornscheuer
Biotechnologically relevant enzymes and proteins


Pyridoxal-5′-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.


Decarboxylase PLP-dependent enzymes Protein-function analysis Racemase 



We thank the European Union (KBBE-2011-5, Grant No. 289350), the DFG (INST 292/118-1 FUGG), and the federal state Mecklenburg-Vorpommern for their financial support. A.M.K. thanks the Deutscher Akademischer Austauschdienst for financial support through the DAAD Study Scholarship. Furthermore, we thank Ina Menyes, Martin Weiss, and Dr. Mark Dörr (all Institute of Biochemistry, Greifswald University) for the analytical support.

Compliance with ethical standards

Conflict of interest

All authors—except HJJ and TvdB as employees of Bioprodict—declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7940_MOESM1_ESM.pdf (791 kb)
ESM 1 (PDF 790 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Anders M. Knight
    • 1
    • 2
  • Alberto Nobili
    • 1
    • 3
    • 4
  • Tom van den Bergh
    • 5
  • Maika Genz
    • 1
  • Henk-Jan Joosten
    • 5
  • Dirk Albrecht
    • 6
  • Katharina Riedel
    • 6
  • Ioannis V. Pavlidis
    • 1
    • 7
  • Uwe T. Bornscheuer
    • 1
  1. 1.Institute of Biochemistry, Department of Biotechnology and Enzyme CatalysisGreifswald UniversityGreifswaldGermany
  2. 2.Division of Biology and BioengineeringCalifornia Institute of TechnologyPasadenaUSA
  3. 3.Department of Cancer Immunology and VirologyDana-Farber Cancer InstituteBostonUSA
  4. 4.Department of Microbiology and ImmunobiologyHarvard Medical SchoolBostonUSA
  5. 5.Bio-ProdictNijmegenthe Netherlands
  6. 6.Institute for Microbiology, Department of Microbial Physiology and Molecular BiologyGreifswald UniversityGreifswaldGermany
  7. 7.Department of BiochemistryUniversity of KasselKasselGermany

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