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Pyridoxal-5′-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for stereoselective amination. D-amino acid transaminases can catalyze stereoselective transamination producing optically pure D-amino acids. The knowledge of substrate binding mode and substrate differentiation mechanism in D-amino acid transaminases comes down to the analysis of the transaminase from <i>Bacillus subtilis</i>. However, at least two groups of D-amino acid transaminases differing in the active site organization are known today. Here, we present a detailed study of D-amino acid transaminase from the gram-negative bacterium <i>Aminobacterium colombiense</i> with a substrate binding mode different from that for the transaminase from <i>B. subtilis</i>. We study the enzyme using kinetic analysis, molecular modeling, and structural analysis of holoenzyme and its complex with D-glutamate. We compare the multipoint binding of D-glutamate with the binding of other substrates, D-aspartate and D-ornithine. QM/MM MD simulation reveals that the substrate can act as a base and its proton can be transferred from the amino group to the α-carboxylate group. This process occurs simultaneously with the nucleophilic attack of the PLP carbon atom by the nitrogen atom of the substrate forming gem-diamine at the transimination step. This explains the absence of the catalytic activity toward (<i>R</i>)-amines that lack an α-carboxylate group. The obtained results clarify another substrate binding mode in D-amino acid transaminases and underpinned the substrate activation mechanism.