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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
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
Azam MA, Jayaram U (2016) Inhibitors of alanine racemase enzyme: a review. J Enzyme Inhib Med Chem 31(4):517–526. doi:10.3109/14756366.2015.1050010
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The Protein Data Bank. Nucleic Acids Res 28(1):235–242
Brückner H, Wittner R, Haasmann S, Langer M, Westhauser T (1994) Liquid chromatographic determination of D- and L-amino acids by derivatization with o-phthaldialdehyde and chiral thiols: applications with reference to biosciences. J Chromatogr A 666:259–273
Eliot AC, Kirsch JF (2004) Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Ann Rev Biochem 73:383–415. doi:10.1146/annurev.biochem.73.011303.074021
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
Eswaramoorthy S, Gerchman S, Graziano V, Kycia H, Studier FW (2003) Structure of a yeast hypothetical protein selected by a structural genomics approach. Acta Cryst Sect D, Biol Cryst 59:127–135
Galkin A, Kulakova L, Yamamoto H, Tanizawa K, Tanaka H, Esaki N, Soda K (1997) Conversion of α-keto acids to D-amino acids by coupling of four enzyme reactions. J Ferment Bioeng 83(3):299–300
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
Höhne M, Schätzle S, Jochens H, Robins K, Bornscheuer UT (2010) Rational assignment of key motifs for function guides in silico enzyme identification. Nature Chem Biol 6(11):807–813. doi:10.1038/nchembio.447
Holt A, Palcic MM (2006) A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat Protocols 1(5):2498–2505. doi:10.1038/nprot.2006.402
Inagaki K, Tanizawa K, Badet B, Walsh CT, Tanaka H, Soda K (1986) Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization. Biochemistry 25(11):3268–3274. doi:10.1021/bi00359a028
Ito T, Iimori J, Takayama S, Moriyama A, Yamauchi A, Hemmi H, Yoshimura T (2013) Conserved pyridoxal protein that regulates Ile and Val metabolism. J Bacteriol 195(24):5439–5449. doi:10.1128/JB.00593-13
Jackson LK, Baldwin J, Akella R, Goldsmith EJ, Phillips MA (2004) Multiple active site conformations revealed by distant site mutation in ornithine decarboxylase. Biochemistry 43(41):12990–12999. doi:10.1021/bi048933l
Jackson LK, Brooks HB, Osterman AL, Goldsmith EJ, Phillips MA (2000) Altering the reaction specificity of eukaryotic ornithine decarboxylase. Biochemistry 39(37):11247–11257
Joosten H-J (2007) 3DM: from data to medicine., PhD thesis, Wageningen University
Ju J, Xu S, Furukawa Y, Zhang Y, Misono H, Minamino T, Namba K, Zhao B, Ohnishi K (2011) Correlation between catalytic activity and monomer-dimer equilibrium of bacterial alanine racemases. J Biochem 149(1):83–89. doi:10.1093/jb/mvq120
Kuipers R, Van Den Bergh T, Joosten HJ, Lekanne dit Deprez RH, Mannens MMAM, Schaap PJ (2010a) Novel tools for extraction and validation of disease-related mutations applied to fabry disease. Hum Mutat 31(9):1026–1032. doi:10.1002/humu.21317
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
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. doi:10.1093/molbev/msw054
Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, USA
Percudani R, Peracchi A (2003) A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO Rep 4(9):850–854. doi:10.1038/sj.embor.embor914
Romero-Romero S, Costas M, Rodriguez-Romero A, Fernandez-Velasco DA (2015) Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins. Phys Chem Chem Phys 17(32):20699–20714. doi:10.1039/C5CP01599E
Rzhetsky A, Nei M (1992) A simple method for estimating and testing minimum-evolution trees. Mol Biol Evol 9(5):945
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425
Schneider G, Käck H, Lindqvist Y (2000) The manifold of vitamin B6 dependent enzymes. Structure 8(1):1–6. doi:10.1016/S0969-2126(00)00085-X
Shaw JP, Petsko GA, Ringe D (1997) Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9-Å resolution. Biochemistry 36(6):1329–1342. doi:10.1021/bi961856c
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7(1):n/a–n/a. doi:10.1038/msb.2011.75
Soda K, Oikawa T, Yokoigawa K (2001) One-pot chemo-enzymatic enantiomerization of racemates. J Mol Catal B Enzym 11:149–153
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 33:566–604
Steffen-Munsberg F, Vickers C, Thontowi A, Schätzle S, Tumlirsch T, Svedendahl Humble M, Land H, Berglund P, Bornscheuer UT, Höhne M (2013) Connecting unexplored protein crystal structures to enzymatic function. ChemCatChem 5:150–153
Sun S, Toney MD (1999) Evidence for a two-base mechanism involving tyrosine-265 from arginine-219 mutants of alanine racemase. Biochemistry 38(13):4058–4065. doi:10.1021/bi982924t
Tatusova T, Ciufo S, Fedorov B, O’Neill K, Tolstoy I (2014) RefSeq microbial genomes database: new representation and annotation strategy. Nucleic Acids Res 42(D1):D553–D559. doi:10.1093/nar/gkt1274
Wu H-M, Kuan Y-C, Chu C-H, Hsu W-H, Wang W-C (2012) Crystal structures of lysine-preferred racemases, the non-antibiotic selectable markers for transgenic plants. PLoS One 7(10):e48301. doi:10.1371/journal.pone.0048301
Zuckerkandl E, Pauling L (1965) Evolutionary divergence and convergence in proteins. In: Bryson V, Vogel HJ (eds) Evolving Genes and Proteins. Academic Press, New York, pp. 97–166
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.
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.
Anders M. Knight and Alberto Nobili contributed equally to this work
Electronic supplementary material
About this article
Cite this article
Knight, A.M., Nobili, A., van den Bergh, T. et al. Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases. Appl Microbiol Biotechnol 101, 1499–1507 (2017). https://doi.org/10.1007/s00253-016-7940-7
- PLP-dependent enzymes
- Protein-function analysis