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Mechanism of D-Cycloserine Inhibition of D-Amino Acid Transaminase from Haliscomenobacter hydrossis

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Abstract

D-cycloserine inhibits pyridoxal-5′-phosphate (PLP)-dependent enzymes. Inhibition effect depend on organization of the active site and mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of the enzyme similarly to the substrate (amino acid), and this interaction is predominantly reversible. Several products of the interaction of PLP with D-cycloserine are known. For some enzymes formation of a stable aromatic product – hydroxyisoxazole-pyridoxamine-5′-phosphate at certain pH – leads to irreversible inhibition. The aim of this work was to study the mechanism of D-cycloserine inhibition of the PLP-dependent D-amino acid transaminase from Haliscomenobacter hydrossis. Spectral methods revealed several products of 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 form of D-cycloserine, and pyridoxamine-5′-phosphate. Formation of hydroxyisoxazole-pyridoxamine-5′-phosphate was not observed. 3D structure of the complex with D-cycloserine was obtained using 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. Ketimine occupied two positions interacting with different active site residues via hydrogen bonds. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and activity of the inhibited transaminase from H. hydrossis could be restored by adding excess of keto substrate or excess of cofactor. The obtained results confirm reversibility of the inhibition by D-cycloserine and interconversion of various adducts of D-cycloserine and PLP.

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Abbreviations

Halhy:

D-amino acid transaminase from the bacterium Haliscomenobacter hydrossis

PLP:

pyridoxal-5′-phosphate

PMP:

pyridoxamine-5′-phosphate

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Funding

This work was supported by the Russian Science Foundation, grant no. 19-14-00164 (in part of kinetic experiments and crystallization of the complex, structure determination and refinement) and by the Ministry of Science and Higher Education of the Russian Federation (in part of spectral investigations). X-ray diffraction experiments were performed with support of the Federal Space Agency of the Russian Federation [project of Space experiments (target works) “Kristallizator”].

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Authors and Affiliations

  1. Bach Institute of Biochemistry, Research Centre of Biotechnology, Russian Academy of Sciences, 119071, Moscow, Russia

    Alina K. Bakunova, Ilya O. Matyuta, Alena Yu. Nikolaeva, Konstantin M. Boyko, Vladimir O. Popov & Ekaterina Yu. Bezsudnova

  2. Kurchatov Complex of NBICS-Technologies, National Research Centre “Kurchatov Institute”, 123182, Moscow, Russia

    Alena Yu. Nikolaeva

  3. Faculty of Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    Vladimir O. Popov

Authors
  1. Alina K. Bakunova
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  2. Ilya O. Matyuta
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  3. Alena Yu. Nikolaeva
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  4. Konstantin M. Boyko
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  5. Vladimir O. Popov
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  6. Ekaterina Yu. Bezsudnova
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Contributions

A.K.B. – planning of experiments; E.Yu.B. and V.O.P. – concept and supervision of the study; A.K.B., A.Yu.N., I.O.M., K.M.B. – conducting experiments; A.K.B., I.O.M., K.M.B., E.Yu.B. – discussion of the results of the study; A.K.B., E.Yu.B. – writing the paper; A.K.B., K.M.B., A.Yu.N., E.Yu.B., V.O.P. – editing of the paper.

Corresponding authors

Correspondence to Alina K. Bakunova or Ekaterina Yu. Bezsudnova.

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The authors declare no conflict of interest in financial or any other sphere. This article does not contain any studies with human participants or animals performed by any of the authors.

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Bakunova, A.K., Matyuta, I.O., Nikolaeva, A.Y. et al. Mechanism of D-Cycloserine Inhibition of D-Amino Acid Transaminase from Haliscomenobacter hydrossis. Biochemistry Moscow 88, 687–697 (2023). https://doi.org/10.1134/S0006297923050115

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