Preprint Article Version 1 This version is not peer-reviewed

Structural Analysis and Substrate Specificity of D-carbamoylase from Pseudomonas

Marina Paronyan
,
HAYKANUSH KOLOYAN
* ,
Hovsep Aganyants
* ,
ARTUR HAMBARDZUMYAN
,
TIGRAN SOGHOMONYAN
,
SONA AVETISYAN
,
SERGEI KOCHAROV
,
HENRY PANOSYAN
,
Vehary Sakanyan
* ,
ANICHKA HOVSEPYAN
Version 1 : Received: 26 August 2024 / Approved: 26 August 2024 / Online: 27 August 2024 (03:15:18 CEST)

How to cite: Paronyan, M.; KOLOYAN, H.; Aganyants, H.; HAMBARDZUMYAN, A.; SOGHOMONYAN, T.; AVETISYAN, S.; KOCHAROV, S.; PANOSYAN, H.; Sakanyan, V.; HOVSEPYAN, A. Structural Analysis and Substrate Specificity of D-carbamoylase from Pseudomonas. Preprints 2024, 2024081865. https://doi.org/10.20944/preprints202408.1865.v1 Paronyan, M.; KOLOYAN, H.; Aganyants, H.; HAMBARDZUMYAN, A.; SOGHOMONYAN, T.; AVETISYAN, S.; KOCHAROV, S.; PANOSYAN, H.; Sakanyan, V.; HOVSEPYAN, A. Structural Analysis and Substrate Specificity of D-carbamoylase from Pseudomonas. Preprints 2024, 2024081865. https://doi.org/10.20944/preprints202408.1865.v1

Abstract

The synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step "hydantoinase process" based on the sequential catalysis of substrates by specific enzymes, D-carbamoylase and D-hydantoinase. In addition to our previously studied D-hydantoinase, we describe here the structural features of D-carbamoylase from Pseudomonas, the encoded gene of which was chemically synthesized and cloned into Escherichia coli. A significant fraction of the overexpressed recombinant protein forms insoluble inclusion bodies, which are partially converted to a soluble state upon treatment with N-lauroylsarcosine or upon incubation of the cells at low temperatures. Analysis of the substrate specificity of the purified His-tag protein showed that D-carbamoylase exhibits the highest activity toward N-carbamoyl-D-alanine and N-carbamoyl-D-tryptophan. Molecular docking analysis revealed the location of the substrate binding site in the three-dimensional structure of D-carbamoylase. Molecular dynamics simulations showed that the binding pocket of the enzyme in complex with N-carbamoyl-D-tryptophan is stabilized within 100 ns. The free energy of binding indicated a possible role of residues Arg176 and Asn173 in the formation of hydrogen bonds between the enzyme and N-carbamoyl-D-tryptophan. The properties of the studied D-carbamoylase and D-hydantoinase support further development of a combined biotechnological process for the production of new drugs and peptide hormones.

Keywords

D-carbamoylase; homology modeling; molecular docking; molecular dynamics; N-carbamoyl-D-alanine; N-carbamoyl-D-tryptophan; inclusion bodies; Pseudomonas

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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