Abstract
d-Amino acids play several key roles and are widely diffused in living organisms, from bacteria (in which d-alanine is a component of the cell wall) to mammals (where d-serine is involved in glutamatergic neurotransmission in the central nervous system). The study of the biological processes involving d-amino acids and their use as clinical or biotechnological biomarkers requires reliable methods of quantifying them. Although “traditional” analytical techniques have been (and still are) employed for such tasks, enzymatic assays based on enzymes which possess a strict stereospecificity (i.e., that are only active on the d-enantiomers of amino acids) allowed the set-up of low-cost protocols with a high sensitivity and selectivity and suitable for determining the d-amino acid content of complex biological samples. The most exploited enzyme in these assays is d-amino acid oxidase, a flavoenzyme that exclusively oxidizes d-amino acids and possesses with a broad substrate specificity and a high kinetic efficiency.
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References
Auclair J L, Patton R L (1950) On the occurrence of D-alanine in the haemolymph of the milkweed bug Oncopeltus fasciatus. Rev Can Biol 9, 3–8.
Corrigan J J (1969) D-amino acids in animals. Science 164, 142–149.
Helfman PM, Bada J L, Shou M Y (1977) Considerations on the role of aspartic acid racemization in the aging process. Gerontology 23, 419–425.
Hashimoto A, Nishikawa T, Oka T et al. (1992) Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. J Chromatogr 582, 41–48.
Snyder S H, Kim P M (2000) D-amino acids as putative neurotransmitters: focus on D-serine. Neurochem Res 25, 553–560.
Oliet S H, Mothet J P (2006) Molecular determinants of D-serine-mediated gliotransmission: from release to function. Glia 54, 726–737.
Pollegioni L, Sacchi S (2010) Metabolism of the neuromodulator D-serine. Cell Mol Life Sci 67, 2387–2404.
Rubio-Barroso S, Santos-Delgado M J, Martin-Olivar C, Polo-Diez L M (2006) Indirect chiral HPLC determination and fluorimetric detection of D-amino acids in milk and oyster samples. J Dairy Sci 89, 82–89.
Friedman M J (1999) Chemistry, nutrition, and microbiology of D-amino acids. Agric Food Chem 47, 3457–3479.
Marchelli R, Palla G, Dossena A et al. (1997) D-ammino acidi: marker molecolari di stagionatura e di tipicità per il Parmigiano-Reggiano e il Grana Padano. Scienza Tecnica Lattiero-Casearia 48, 21–32.
Caligiuri, A., D’Arrigo, P., Rosini, E. et al. (2006) Enzymatic conversion of unnatural amino acids by yeast D-amino acid oxidase. Adv Synth Catal 348, 2183–2190.
Pollegioni L., Piubelli L., Sacchi S et al. (2007) Physiological functions of D-amino acid oxidases: from yeast to humans. Cell Mol Life Sci 64, 1373–1394.
Sacchi S, Lorenzi S, Molla G et al. (2002) Engineering the substrate specificity of D-amino-acid oxidase. J Biol Chem 277, 27510–27516.
Sacchi S, Rosini E, Molla G et al. (2004) Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution. Protein Eng Des Sel 17, 517–25.
Oguri S, Nomura M, Fujita Y (2005) A new strategy for the selective determination of D-amino acids: enzymatic and chemical modifications for pre-column derivatization. J Chromatogr A 1078, 51–58.
Rosini E, Molla G, Rossetti C et al. (2008) A biosensor for all D-amino acids using evolved D-amino acid oxidase. J Biotechnol 135, 377–384.
Pernot P, Mothet J P, Schuvailo O et al. (2008) Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system. Anal Chem 80, 1589–1597.
Molla G, Vegezzi C, Pilone MS, Pollegioni L (1998) Overexpression in Escherichia coli of a recombinant chimeric Rhodotorula gracilis D-amino acid oxidase. Protein Expr Purif 14, 289–294.
Fantinato S, Pollegioni L, Pilone M S (2001) Engineering, expression and purification of a His-tagged chimeric D-amino acid oxidase from Rhodotorula gracilis. Enzyme Microb Technol 29, 407–412.
Negri A, Massey V, Williams C H Jr (1987) D-Aspartate oxidase from beef kidney: purification and properties. J Biol Chem 262, 10026–10034.
Simonic T, Duga S, Negri A et al. (1997) cDNA cloning and expression of the flavoprotein D-aspartate oxidase from bovine kidney cortex. Bioch J 322, 729–735.
Bohme A, Winkler O (1954) Zur Bestimmung geringer Mengen Acetaldehyd. Z Anal Chem 412, 1–5.
Walsh C T, Schonbrunn A, Abeles R H (1971) Studies on the mechanism of action of D-amino acid oxidase: evidence for removal of substrate α-hydrogen as a proton. J Biol Chem 216, 6855–6866.
Chlumsky L J, Zhang L, Rhamsey A J, Schuman Jorns M (1993) Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine. Biochemistry 32, 11132–11142.
Mori N, Sano M, Tani Y, Yamada H (1980) Purification and properties of sarcosine oxidase from Cylindrocarpon didymum M-1. Agric Biol Chem 44, 1391–1397.
Pollegioni L, Sacchi S, Caldinelli L et al. (2007) Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach. Curr Protein Pept Sci 8, 600–618.
Molla G, Sacchi S, Bernasconi M et al. (2006) Characterization of human D-amino acid oxidase. FEBS Lett 580, 2358–2364.
Pollegioni L, Caldinelli L, Molla G et al. (2004) Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources. Biotechnol Prog 20, 467–473.
Acknowledgments
This work was supported by grants from Fondo di Ateneo per la Ricerca (University of Insubria) to G. Molla and L. Piubelli.
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Molla, G., Piubelli, L., Volontè, F., Pilone, M.S. (2012). Enzymatic Detection of d-Amino Acids. In: Pollegioni, L., Servi, S. (eds) Unnatural Amino Acids. Methods in Molecular Biology, vol 794. Humana Press. https://doi.org/10.1007/978-1-61779-331-8_18
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DOI: https://doi.org/10.1007/978-1-61779-331-8_18
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