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Characterization of proteins from the 3N5M family reveals an operationally stable amine transaminase
(2022)
Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the β-alanine:pyruvate transaminase cluster (3N5M) — a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1: Bilophilia wadsworthia, TA-5: Halomonas elongata, TA-9: Burkholderia cepacia, and TA-10: Burkholderia multivorans) were obtained in a soluble form as tetramers in E. coli. During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from Burkholderia multivorans features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C.
Abstract
Amine transaminases (ATAs) are biocatalysts for the synthesis of chiral amines and can be identified in sequence databases by specific sequence motifs. This study shows that the activity level towards the model substrate 1‐phenylethylamine can be predicted solely from the sequence. To demonstrate this, 15 putative ATAs with a different distribution of hydrophobic or hydrophilic amino acid side chains near the active site were characterized. Hydrophobic side chains were associated with a high activity level and were a better predictor of activity than global sequence identity to known ATAs with high or low activities. Enzyme stability investigations revealed that four out of the 15 ATAs showed a good operational stability.
Promiscuous Dehalogenase Activity of the Epoxide Hydrolase CorEH from Corynebacterium sp. C12
(2021)
Haloalkane dehalogenases and epoxide hydrolases are phylogenetically related and structurally homologous enzymes that use nucleophilic aspartate residues for an SN2 attack on their substrates. Despite their mechanistic similarities, no enzymes are known that exhibit both epoxide hydrolase and dehalogenase activity. We screened a subset of epoxide hydrolases, closely related to dehalogenases, for dehalogenase activity and found that the epoxide hydrolase CorEH from Corynebacterium sp. C12 exhibits promiscuous dehalogenase activity. Compared to the hydrolysis of epoxides like cyclohexene oxide (1.41 μmol min–1 mg–1), the dehalogenation of haloalkanes like 1-bromobutane (0.25 nmol min–1 mg–1) is about 5000-fold lower. In addition to the activity with 1-bromobutane, dehalogenase activity was detected with other substrates like 1-bromohexane, 1,2-dibromoethane, 1-iodobutane, and 1-iodohexane. This study shows that dual epoxide hydrolase and dehalogenase activity can be present in one naturally occurring protein scaffold.
Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades
(2019)
Plant-derived carbohydrates are an abundant renewable re- source. Transformation of carbohydrates into new products, in- cluding amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, bio- catalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step bio- catalysis was performed to functionalize d-galactose-contain- ing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from
Agaricus bisporus followed by the w-transaminase from Chro- mobacterium violaceum (Cvi-w-TA). Formation of 6-amino-6- deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino- 2-deoxy-6-aldo-d-galactose was confirmed by mass spectrome- try. The activity of Cvi-w-TA was highest towards 6-aldo-d-gal- actose, for which the highest yield of 6-amino-6-deoxy-d-galac- tose (67%) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6- aldo-d-galactose.