8AQ0

Crystal structure of L-N-Carbamoylase from Sinorhizobium meliloti mutant L217G/F329C

Summary for 8AQ0

• Entry DOI: 10.2210/pdb8aq0/pdb
• Related: 8APZ
• Descriptor: N-carbamoyl-L-amino-acid hydrolase, ZINC ION, FE (III) ION, ... (6 entities in total)
• Functional Keywords: peptidase family m20/m25/m40, hydrolase
• Biological source: Sinorhizobium meliloti
• Total number of polymer chains: 2
• Total formula weight: 94847.11

Authors

Rozeboom, H.J.,Mayer, C. (deposition date: 2022-08-11, release date: 2022-11-16, Last modification date: 2024-05-01)

Primary citation

Rubini, R.,Jansen, S.C.,Beekhuis, H.,Rozeboom, H.J.,Mayer, C.
Selecting Better Biocatalysts by Complementing Recoded Bacteria.
Angew.Chem.Int.Ed.Engl., 62:e202213942-e202213942, 2023

PubMed Abstract: In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild-type enzyme for ncAA-precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts.

PubMed: 36342942
DOI: 10.1002/anie.202213942

Experimental method

X-RAY DIFFRACTION (2.3 Å)