Quantum Chemical Study of Dual-Substrate Recognition in ω-Transaminase

Abstract

ω-Transaminases are attractive biocatalysts for the production of chiral amines. These enzymes usually have a broad substrate range. Their substrates include hydrophobic amines as well as amino acids, a feature referred to as dual-substrate recognition. In the present study, the reaction mechanism for the half-transamination of l-alanine to pyruvate in (S)-selective Chromobacterium violaceum ω-transaminase is investigated using density functional theory calculations. The role of a flexible arginine residue, Arg416, in the dual-substrate recognition is investigated by employing two active-site models, one including this residue and one lacking it. The results of this study are compared to those of the mechanism of the conversion of (S)-1-phenylethylamine to acetophenone. The calculations suggest that the deaminations of amino acids and hydrophobic amines follow essentially the same mechanism, but the energetics of the reactions differ significantly. It is shown that the amine is kinetically favored in the half-transamination of l-alanine/pyruvate, whereas the ketone is kinetically favored in the half-transamination of (S)-1-phenylethylamine/acetophenone. The calculations further support the proposal that the arginine residue facilitates the dual-substrate recognition by functioning as an arginine switch, where the side chain is positioned inside or outside of the active site depending on the substrate. Arg416 participates in the binding of l-alanine by forming a salt bridge to the carboxylate moiety, whereas the conversion of (S)-1-phenylethylamine is feasible in the absence of Arg416, which here represents the case in which the side chain of Arg416 is positioned outside of the active site.

Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades

“…The R416 (flipping arginine) side chain was turned slightly upwards away from the PMP to gain space in the large binding pocket, since the sugar substrates did not contain charged residues. [80,81] The docking ligands 2b (6-aldo-d-galactose) and the corresponding 6-aldo-d-galactosyl-containing aldo-lactose and aldo-raffinose were constructed with the built-in oligosaccharide building tool of YASARA, by using the b-d-conformation of each sugar.T he docking ligand aldo-melibiose was constructed by oxidizing the corresponding melibiose structure (PubChem Identifier:C ID 11458). The XLLG molecule ( Figure S6) was extracted as a ligand from the crystal structure PDB ID:2 VH9 and subsequently oxidized to obtain aldo-XLLG.…”

The crystal structure of the tetrameric DABA‐aminotransferase EctB, a rate‐limiting enzyme in the ectoine biosynthesis pathway

“…Class III x-TAs, such as the herein presented EctB, must have dual substrate recognition mechanisms to differentiate between two substrate pairs [39,40] and be able to specifically catalyze the transfer of the distal amine group. Several studies show that the active site of many aminotransferases has two binding pockets, denoted according to their relative position to the PLP coenzyme (Figs 1C and 2), and that the orientation of substrates in the active site is often coordinated by one or two arginine residues that form strong directional bonds to the substrate's carboxylate group [39,[41][42][43][44][45][46] by a so-called end-on geometry [47]. When the arginine-carboxylate bond is not required, some aminotransferases neutralize the arginine through a strong ionic bond with a glutamate residue (i.e., glutamate switch), while others have a flexible arginine that moves in and out of the active site (i.e., arginine switch; Fig.…”

Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids

“…In the current study, replacing arginine with another positively charged, but less bulky amino acid lysine, generated an enzyme, which retained less than 3% relative activity with all tested substrates, whether they were acids or esters (Figs 3 and 4 ). The side chain of the R416 residue is positioned inside or outside of the active site depending on the substrate 32 . If the substrate contains a carboxylic group then residue R416 forms a salt bridge with the carboxylate moiety, therefore facilitating deamination by decreasing the energy barrier 32 .…”

“…The side chain of the R416 residue is positioned inside or outside of the active site depending on the substrate 32 . If the substrate contains a carboxylic group then residue R416 forms a salt bridge with the carboxylate moiety, therefore facilitating deamination by decreasing the energy barrier 32 . If a substrate without a carboxylate moiety is used, the R416 side chain faces outwards 32 .…”

“…If the substrate contains a carboxylic group then residue R416 forms a salt bridge with the carboxylate moiety, therefore facilitating deamination by decreasing the energy barrier 32 . If a substrate without a carboxylate moiety is used, the R416 side chain faces outwards 32 . However, this seems to be a specific case with the CV_TA, since when the arginine residue of ω-TA of V .…”