title: Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases.
abstract: 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.
authors: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany.|Division of Biology and Bioengineering, California Institute of Technology, 1200 E. California Blvd. MC 210-41, Pasadena, CA, 91125, USA.
authors: Anders M
authors: AM
authors: Knight
authors: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany.|Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, 02115, USA.|Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA.
authors: Alberto
authors: A
authors: Nobili
authors: Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA, Nijmegen, the Netherlands.
authors: Tom
authors: T
authors: van den Bergh
authors: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany.
authors: Maika
authors: M
authors: Genz
authors: Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA, Nijmegen, the Netherlands.
authors: Henk-Jan
authors: HJ
authors: Joosten
authors: Institute for Microbiology, Department of Microbial Physiology and Molecular Biology, Greifswald University, Friedrich-Ludwig-Jahn-Str. 15, 17487, Greifswald, Germany.
authors: Dirk
authors: D
authors: Albrecht
authors: Institute for Microbiology, Department of Microbial Physiology and Molecular Biology, Greifswald University, Friedrich-Ludwig-Jahn-Str. 15, 17487, Greifswald, Germany.
authors: Katharina
authors: K
authors: Riedel
authors: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany.|Department of Biochemistry, University of Kassel, Heinrich-Plett-Str. 40, D-34132, Kassel, Germany.
authors: Ioannis V
authors: IV
authors: Pavlidis
authors: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany. uwe.bornscheuer@uni-greifswald.de.
authors: Uwe T
authors: UT
authors: Bornscheuer
mesh_terms: D002262:Carboxy-Lyases / Q000737:chemistry / Q000378:metabolism; D019295:Computational Biology / Q000379:methods*; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011732:Pyridoxal Phosphate; D002262:Carboxy-Lyases; D019998:Racemases and Epimerases
keywords: Decarboxylase; PLP-dependent enzymes; Protein-function analysis; Racemase