Mechanism of D-cycloserine inhibition of D-amino acid transaminase from <i>Haliscomenobacter hydrossis</i>

Abstract

D-cycloserine inhibits pyridoxal-5′-phosphate (PLP)-dependent enzymes. The inhibition efficiency depends on the organization of their active center and the mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of enzyme similarly to substrate amino donor, and the interaction is predominantly reversible. Inhibition products include hydroxyisoxazole-pyridoxamine-5′-phosphate, oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5′-phosphate and cyclic or open forms of D-cycloserine, pyridoxamine-5′-phosphate, etc. For some enzymes the formation of a stable aromatic product - hydroxyisoxazole can lead to irreversible D-cycloserine inhibition at certain pH value. The aim of this work was to study the mechanism of D-cycloserine inhibition of PLP-dependent D-amino acid transaminase from the bacterium Haliscomenobacter hydrossis. Spectral methods revealed several products of the interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5′-phosphate and cyclic or open forms of D-cycloserine, pyridoxamine-5′-phosphate. The formation of hydroxyisoxazole-pyridoxamine-5′-phosphate was not observed. The 3D structure of the complex of transaminase with D-cycloserine was obtained by X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5′-phosphate and D-cycloserine in the cyclic form was found; the ketimine occupied two positions and was coordinated via hydrogen bonds with different active site residues. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and the activity of transaminase from H. hydrossis can be restored by adding an excess of keto substrate as well as by adding an excess of cofactor. The results obtained confirm the reversibility of D-cycloserine inhibition and the conversion of various adducts of D-cycloserine and PLP into each other.