The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Abstract

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

Development of an aminotransferase‐driven biocatalytic cascade for deracemization of d,l‐phosphinothricin

“…The Ym d ‐AAT, Td DDO, and Gb CAT were considered for coexpression in E. coli BL21(DE3) using plasmid pET28a. As well known, d ‐Glu is an essential component of bacterial cell wall peptidoglycan (Bakunova et al, 2021; Johnson et al, 2013). As shown in Table 2, Td DDO exhibited high activity (46.3 U/mg at 37°C) and excellent affinity ( K m d ‐Glu = 2.16 mM) toward d ‐Glu, the basal expression of Td DDO would result in the death of E. coli before induction if E. coli BL21(DE3) were used as host (Takahashi et al, 2019).…”

“…The Ym D-AAT, Td DDO, and GbCAT were considered for coexpression in E. coli BL21(DE3) using plasmid pET28a. As well known, D-Glu is an essential component of bacterial cell wall peptidoglycan (Bakunova et al, 2021;Johnson et al, 2013).…”

Nutrición animal: Texto de formación universitaria

“…Además, la vitamina B6 puede extinguir las especies reactivas de oxígeno (ROS), por lo que es esencial para la síntesis de clorofila y la fotosíntesis (Parra et al, 2018). Las reacciones de transaminación son catalizadas por transaminasas específicas (también llamadas aminotransferasas), que requieren piridoxal fosfato como coenzima (Bakunova et al, 2021). Las transaminasas (aminotransferasas) están ampliamente distribuidas en los tejidos y son especialmente activas en el músculo cardíaco, el hígado, el músculo esquelético y los riñones.…”

Interaction of D-cycloserine with a D-amino acid transaminase from Haliscomenobacter hydrossis

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Motivation and Aim: Studying the structures of enzyme complexes with inhibitors is a great way to explore the substrate recognition mode of the enzyme. Recently, we characterized pyridoxal-5'-phosphate (PLP)-dependent D-amino acid transaminase (DAAT) from bacterium Haliscomenobacter hydrossis (Halhy) with a new type of the recognition site for the substrate α-carboxyl group [1]. D-cycloserine (D-CS) is a cyclic analog of serine.

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