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

Alina K Bakunova,Alena Yu Nikolaeva,Ekaterina Yu Bezsudnova,Maria G Khrenova,Tatiana V Rakitina,Vladimir O Popov,Tatiana Y Isaikina,Konstantin M Boyko

doi:10.3390/molecules26165053

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.

Highlights

The superfamily of pyridoxal-50 -phosphate (PLP)-dependent transaminases (TAs; aminotransferases; EC 2.6.1.) is an excellent basis for studying sequence-structure-function relationships in enzymes: TAs exhibit a diversity of substrate specificity and activity using a uniform structural scaffold
Searching for sequences homologous to the sequence of TA from C. pusillum within the complete genome of H. hydrossis revealed a sequence of TA of PLP fold type IV
The sequence of the putative transaminase contained unique specificity-determining sequence motifs, which differ from the sequence motifs of the canonical TAs of PLP fold type IV (Table 1)

Summary

Introduction

The superfamily of pyridoxal-50 -phosphate (PLP)-dependent transaminases (TAs; aminotransferases; EC 2.6.1.) is an excellent basis for studying sequence-structure-function relationships in enzymes: TAs exhibit a diversity of substrate specificity and activity using a uniform structural scaffold. The transamination reaction proceeds via a double displacement reversible mechanism; one catalytic turnover includes two half-reactions with the conversion of covalently bound cofactor PLP to free pyridoxamine 5’-phosphate (PMP) and release of the first product and subsequent recovery of the PLP form of cofactor accompanying by transfer of the amino group to a keto substrate and the release of a new amino compound [1,2,3]. Strict (R)-selectivity or (S)-selectivity of TAs of PLP fold type IV is implemented within geometrically similar active sites and results from the different amino acid compositions The superfamily of PLP-dependent TAs of fold IV (a superfamily of D-amino acid aminotransferases) includes three canonical families: D-amino acid TAs (DAATs, EC 2.6.1.21), branched-chain

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