Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases
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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.
KeywordsDecarboxylase 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.
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Conflict of interest
All authors—except HJJ and TvdB as employees of Bioprodict—declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Anthony KG, Strych U, Yeung KR, Shoen CS, Perez O, Krause KL, Cynamon MH, Aristoff PA, Koski RA (2011) New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis. PLoS One 6(5):e20374. doi: 10.1371/journal.pone.0020374 CrossRefPubMedPubMedCentralGoogle Scholar
- Espaillat A, Carrasco-López C, Bernardo-García N, Pietrosemoli N, Otero LH, Álvarez L, de Pedro MA, Pazos F, Davis BM, Waldor MK, Hermoso JA, Cava F (2014) Structural basis for the broad specificity of a new family of amino-acid racemases. Acta Cryst Sect D, Biol Cryst 70(Pt 1):79–90. doi: 10.1107/S1399004713024838 CrossRefGoogle Scholar
- Henke E, Pleiss J, Bornscheuer UT (2002) Activity of lipases and esterases towards tertiary alcohols: insights into structure-function relationships. Angew Chem Int Ed 41(17):3211–3213. doi: 10.1002/1521-3773(20020902)41:17<3211::AID-ANIE3211>3.0.CO;2-U CrossRefGoogle Scholar
- Joosten H-J (2007) 3DM: from data to medicine., PhD thesis, Wageningen UniversityGoogle Scholar
- Kuipers RK, Joosten H-J, van Berkel WJH, Leferink NGH, Rooijen E, Ittmann E, van Zimmeren F, Jochens H, Bornscheuer UT, Vriend G, dos Santos VAPM, Schaap PJ (2010b) 3DM: systematic analysis of heterogeneous superfamily data to discover protein functionalities. Proteins 78(9):2101–2113. doi: 10.1002/prot.22725 PubMedGoogle Scholar
- Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, USAGoogle Scholar
- Rzhetsky A, Nei M (1992) A simple method for estimating and testing minimum-evolution trees. Mol Biol Evol 9(5):945Google Scholar
- Steffen-Munsberg F, Vickers C, Kohls H, Land H, Mallin H, Nobili A, Skalden L, van den Bergh T, Joosten H-J, Berglund P, Höhne M, Bornscheuer UT (2015) Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications. Biotechnol Adv 33:566–604CrossRefPubMedGoogle Scholar