Structural Analysis and Substrate Specificity of D-Carbamoylase from Pseudomonas.

Paronyan, Marina; Koloyan, Haykanush; Aganyants, Hovsep; Hambardzumyan, Artur; Soghomonyan, Tigran; Avetisyan, Sona; Kocharov, Sergey; Panosyan, Henry; Sakanyan, Vehary; Hovsepyan, Anichka

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. Here, we describe 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 cells at 28 °C. Purified His-tagged protein exhibits the highest activity towards N-carbamoyl-D-alanine and N-carbamoyl-D-tryptophan. Comprehensive virtual analysis of the interactions of bulky carbamylated amino acids with D-carbamoylase provided valuable information. 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 was stabilized within 100 nanoseconds. The free energy data showed that Arg176 and Asn173 formed hydrogen bonds between the enzyme and substrates. The studies of D-carbamoylases and the properties of our previously obtained D-hydantoinase suggest the possibility of developing a harmonized biotechnological process for the production of new drugs and peptide hormones.

MULTIENZYME complexes; BIOTECHNOLOGY; BIOCHEMICAL substrates; MOLECULAR docking; RECOMBINANT proteins

BioTech, 2024, Vol 13, Issue 4, p40

2673-6284

Academic Journal

10.3390/biotech13040040