Structure and Assembly of the Proline Reductase Complex from Clostridioides difficile

The pathogenic bacterium Clostridioides difficile has become a significant healthcare concern. The pathogenicity of this bacterium is connected to its highly adaptive metabolism. This study aimed to elucidate the structure and assembly of the D-proline reductase complex (Prd), a pivotal enzyme in Stickland fermentation, comprising the pyruvoyl-dependent PrdA and the selenocysteine-containing PrdB. Throughout this research, a novel enzyme mediating the maturation of PrdA was discovered and named PrdH. It was demonstrated that this maturation requires the PrdB subunit, despite the ability of PrdH to interact solely with PrdA. Detailed analysis of this processing reaction revealed the enzymatic nature of PrdH. Successful in vitro catalysis of D-proline reduction was achieved by incorporating selenocysteine into recombinantly expressed PrdB, producing a functional enzyme. Mutagenesis of PrdA and PrdH elucidated critical amino acids and revealed interaction dynamics, notably identifying arginine at position 32 in PrdH as crucial for both interaction with PrdA and mediation of the processing reaction. The interaction between PrdA and PrdH was found to be transient, with PrdH detaching after the processing reaction. However, an active site mutant of PrdA facilitated the capture of a bound PrdA/PrdH complex, offering prospects for future structural studies. The crystal structure of PrdD/PrdE, two pseudoenzymes resembling the catalytic domain of PrdA, was solved, providing initial structural insights into a Prd-related protein. To further investigate the D-proline reductase, challenges related to denaturation and aggregation of full length PrdA during recombinant expression were overcome through co-expression with prdB and prdH. This approach suggested that the maturation of PrdA is a prerequisite for correct assembly of the complex. The recombinantly expressed complex was suitable for further structural studies through negative-stain TEM and single particle analysis via cryo-EM, leading to the resolution of a processed, heterohexameric PrdA/PrdB complex. This work has illuminated various structural and functional aspects of the D-proline reductase complex, revealing it as an intricately dynamic system. By unraveling its maturation and assembly processes, this study contributes to a deeper understanding of this critical enzyme in the metabolism of C. difficile.