Structural Analysis and Substrate Specificity of D-carbamoylase from <em>Pseudomonas</em>

The synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step &quot;hydantoinase process&quot; 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.