by Alina K. Bakunova | Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian
Academy of Sciences, Moscow, Russia
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. D-cycloserine (D-CS) is a cyclic analog of serine. Various PLPdependent enzymes, including transaminases (TA), are able to accommodate D-CS in their
active sites. D-CS is proposed to react with TA through the canonical sequence of reaction
steps and finally forms irreversibly a stable aromatic compound with PLP or dissociates
from the active site. Determination of the structure of the Halhy complex with D-CS can
shed light on both the mechanism of D-CS interaction with Halhy and the mode of substrate
binding in the active site of the new type of DAAT.
Methods and Algorithms:
We explored the interaction of D-CS with Halhy by the combination of UV-Vis spectroscopy,
enzyme kinetics and X-ray crystallography.
Results:
D-CS is a strong inhibitor of Halhy with IC50 value of 3 μM. The inhibited enzyme shows an
absorbance maximum near 330-340 nm, indicative of a completed catalytic half-reaction. In
the crystal structure D-CS is covalently attached to the cofactor forming a ketimine
intermediate an intermediate product. The ketimine occupies two position. Contrary to
expectations, D-CS is not held by numerous noncovalent interactions in the active site. The
analog of a-COOH group is coordinated via Lys241 and the catalytic Lys143.
Conclusion:
D-CS binds PLP irreversibly; however, the excess of PLP restores the activity of Halhy. via
substitution of PLP-D-CS adduct in the active site. In the active site of Halhy D-CS interacts
specifically with PLP, however, no specific interaction with the residues of the recognition
site is observed.