Structural Determinants of the Stereoinverting Activity of Pseudomonas stutzeri d-Phenylglycine Aminotransferase.

Aromatic d-amino acids are key precursors for the production of many small molecule therapeutics. Therefore, the development of biocatalytic methods for their synthesis is of great interest. An enzyme that has great potential as a biocatalyst for the synthesis of d-amino acids is the stereoinverting d-phenylglycine aminotransferase (DPAT) from Pseudomonas stutzeri ST-201. This enzyme catalyzes a unique l to d transamination reaction that produces d-phenylglycine and α-ketoglutarate from benzoylformate and l-glutamate, via a mechanism that is poorly understood. Here, we present the crystal structure of DPAT, which shows that the enzyme folds into a two-domain structure representative of class III aminotransferases. Guided by the crystal structure, we performed saturation mutagenesis to probe the substrate binding pockets of the enzyme. These experiments helped us identify two arginine residues (R34 and R407), one in each binding pocket, that are essential to catalysis. Together with kinetic analyses using a library of amino acid substrates, our mutagenesis and structural studies allow us to propose a binding model that explains the dual l/d specificity of DPAT. Our kinetic analyses also demonstrate that DPAT can catalyze the transamination of ß- and γ-amino acids, reclassifying this enzyme as an ω-aminotransferase. Collectively, our studies highlight that the DPAT active site is amenable to protein engineering for expansion of its substrate scope, which offers the opportunity to generate new biocatalysts for the synthesis of a variety of valuable optically pure d-amino acids from inexpensive and abundant l-amino acids.

Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity.

D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent KM and kcat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in kcat/KM relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.

A high-throughput assay for screening L- or D-amino acid specific aminotransferase mutant libraries.

Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the L-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either L- or D-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 D-amino acid aminotransferase displaying a decrease in k(cat)/K(M) of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate L-leucine than L-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering.