Antranikian, G. | bioRxiv Science

bioRxiv2023

Structural and biochemical characterisation of the N-Carbamoyl-beta-Alanine Amidohydrolases from Rhizobium radiobacter MDC 8606

N-Carbamoyl-{beta}-Alanine Amidohydrolase (C{beta}AA) constitute one of the most important groups of industrially relevant enzymes used in production of optically pure amino acids and derivatives. In this study, a N-carbamoyl-{beta}-alanine amidohydrolase encoding gene from Rhizobium radiobacter MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrC{beta}AA) showed a specific activity of 14 U/mg using N-carbamoyl-{beta}-alanine as a substrate with an optimum activity of 55{degrees}C at pH 8.0. In this work, we report also the first prokaryotic N-carbamoyl-{beta}-alanine amidohydrolases structure at a resolution of 2.0 [A]. A discontinuous catalytic domain and a dimerization domain attached through a flexible hinge region at the domain interface has been revealed. We have found that the ligand is interacting with a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291) residues. Studies let us to explain the preference on the enzyme for linear carbamoyl substrates as large carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrC{beta}AA from the Rhizobium radiobacter MDC 8606 for the industrial production of L--, L-{beta}-, and L-{gamma} - amino acids. The structural analysis provides new insights on enzyme-substrate interaction, which shed light on engineering of N-carbamoyl-{beta}-alanine amidohydrolases for high catalytic activity and broad substrate specificity.

biochemistry↗

bioRxiv2022

Structure-based discovery of a non-canonical prototype long-chain monoacylglycerol lipase through an endogenous catalysis intermediate complex

The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzymes active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.

biochemistry↗