Activity of yeast d-amino acid oxidase on aromatic unnatural amino acids
Introduction
Unnatural amino acids are a growing group of compounds required for a number of biotechnological applications (from the pharmaceuticals and cosmetics to the agrochemicals field). Furthermore, synthetic α-amino acids are used as chiral building blocks and molecular scaffolds in constructing chemical combinatorial libraries [1], for the de novo design of peptides [2], [3], and as valuable pharmaceuticals (in their own right or as components of numerous therapeutically relevant compounds) [4] and they are becoming crucial tools for modern drug discovery. The worldwide increase of the market for enantiopure raw materials, largely required to support the development of new pharmaceutical and agrochemical products, is also giving an increase in the use of biocatalysts for their production. Particularly, the market for amino acids in synthesis shows an annual rate of 5–7% [3]. In recent years, new chiral technologies and improved enantioselective processes have been developed [5], [6]. We recently provided an example for the combination of biological tools for the production of enantiopure unnatural l-amino acids, both using a single enzyme [7] and a multienzymatic system [8]. In both cases we employed the FAD-containing flavoenzyme d-amino acid oxidase (EC 1.4.3.3, DAAO) from the yeast Rhodotorula gracilis (RgDAAO). DAAO catalyses the dehydrogenation of the d-isomer of amino acids to give the corresponding α-keto acids, ammonia and hydrogen peroxide. The overall reaction is shown in Eqs. (1a), (1b), (1c):E ∼ FADox + d-amino acid ↔ E ∼ FADred + imino acidimino acid + H2O → α-keto acid + NH3E ∼ FADred + O2 → E ∼ FADox + H2O2RgDAAO is highly stereoselective (l-amino acids are neither substrates nor inhibitors), it exhibits a very high turnover number, tight binding with the coenzyme FAD, and a broad substrate specificity (for a review see [9]). These properties make RgDAAO a suitable enzyme for biotechnological applications, e.g. for the two-step conversion of cephalosporin C into 7-aminocephalosporanic acid, to detect and quantify d-amino acids, to produce α-keto acids from essential d-amino acids, and to resolve racemic mixtures of d,l-amino acids (for a review see [10]). Furthermore, the solution of the 3D structure of RgDAAO in complex with different substrate analogues and ligands at high resolution [11], [12] allowed a deep investigation of its structure–function relationships and the rational re-design of its substrate specificity [12], [13], [7].
Here, we continue on the use of DAAO as a biological tool for the deracemization of racemic materials, and in particular we studied the enzymatic activity of wild-type and M213G RgDAAOs towards racemic mixtures of unnatural aromatic amino acids to obtain the resolution of the corresponding l-amino acid component.
Section snippets
Materials
The pure d-isomer of 1- and 2-naphthyl-alanine was purchased from Sigma–Aldrich and Bachem, respectively; d-phenylglycine, d-phenylalanine and d-4-OH-phenylglycine were purchased from Fluka; the d-isomer of 1- and 2-naphthyl-glycine was isolated by chiral HPLC chromatography as detailed in [7]. d-Homo-phenylalanine was obtained by subtilisin catalysed resolution of rac-N-Boc-homo-phenylalanine methyl ester.
Enzymes expression and purification
Recombinant wild-type and M213G RgDAAOs were expressed in BL21(DE3)pLysS Escherichia coli
Kinetic properties of wild-type RgDAAO on aromatic d-amino acids
DAAO from the yeast R. gracilis shows a broad substrate specificity: nonpolar and aromatic d-amino acids appeared to be the best substrates [16]. For aromatic natural amino acids the wild-type DAAO showed with phenylalanine a lower Km,app value than that determined for the reference substrate d-alanine (0.3 mM vs. 0.8 mM, respectively). Moreover Vmax,app value similar respect to d-alanine (≈4000 min−1 at pH 8.5, 21% oxygen saturation and 25 °C) [16]. Table 1 summarizes the apparent kinetic
Conclusion
We have produced two single-point mutant enzymes using a structure-based rational design approach with an enlarged substrate specificity: the M213R mutant RgDAAO active on acidic d-amino acids [13] and the M213G mutant RgDAAO more active and unnatural naphthyl-amino acids [7]. A comparison of the active site of wild-type RgDAAO and of the model obtained for the M213G and M213R mutants proteins is depicted in Fig. 2.
In the present work, we analysed the substrate specificity of wild-type and
Acknowledgements
This work was supported by a grant from FAR to LP and GM. Financial support from COFIN-MURST to SS is acknowledged. The work is a collaboration within the COST Action D 25 Stereoselective and environmentally friendly reactions catalysed by enzymes, WG 6.
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