Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH

Abstract

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.

Keywords: Clostridioides difficile; Stickland metabolism; autoproteolysis; proline reductase; pyruvoyl enzyme.

Fig. 1.

Proline reductase Prd is a pyruvoyl- and selenocysteine-containing enzyme. A) Reduction of D-proline starts with binding the substrate at the pyruvoyl cofactor of the α-subunit of PrdA. The selenocysteine of PrdB attacks the α-C-atom of D-proline, which induces ring cleavage and subsequent release of the product 5-aminovalerate. Regeneration of the complex is mediated by the electron transport protein PrdC (5). B) Organization of the prd-operon in Clostridioides difficile. PrdR regulates the expression of the following genes in response to the cellular D-proline level. PrdA and PrdB encode the two subunits of the proline reductase complex. PrdD and PrdE/E2 share high structural similarities with PrdA, but their function is still unknown. PrdF encodes a proline racemase, catalyzing the conversion of L-proline to D-proline. The enzyme CD630_32430 (PrdH) catalyzes pyruvoyl cofactor formation in PrdA and is the main focus of this study. C) Proposed mechanism of pyruvoyl cofactor formation in PrdA, based on observations made for pyruvoyl-dependent decarboxylases (7).

Fig. 2.

Unprocessed PrdA interacts with PrdB. A) SDS-PAGE analysis of recombinant PrdA_149-626 (1) and PrdB_U151C (2). B) SEC-MALS results of three independently performed experiments. PrdA_149-626 (eluting at approx. 11.8 mL) elutes with a single peak. The measured mass of ∼80 kDa indicates a rapid equilibrium between monomeric and dimeric states. The measured mass of PrdB_U151C (∼24 kDa, eluting at 15.5 mL) is consistent with the calculated mass of the monomer. The mixture of PrdA_149-626 and PrdB_U151C (eluting at 11.5 mL) results in a shifted elution with a measured mass of ∼125 kDa, indicating strong interaction between the two subunits. Similar to PrdA_149-626, a rapid equilibrium between different oligomeric states is assumed. C) SEC-MALS analysis of a mixture of PrdA_149-584 and PrdB_U151C. No interaction between the two proteins is observed.

Fig. 3.

The homodimeric protein PrdH catalyzes cleavage of PrdA in the PrdA/B complex. A) SEC-MALS analysis of recombinant PrdH reveals a dimeric protein. B) The structure of the PrdH dimer as predicted with Alphafold2 via ColabFold (17). Colors represent the pLDDT confidence score of AlphaFold2 (blue = highly confident, red = poorly confident). The image was created using PyMol (18). C) Time-dependent in vitro processing of π-PrdA fragment PrdA_149-626 in complex with PrdB by PrdH at equimolar ratio. D) Band intensities were measured using ImageJ (19). The amount of processed PrdA is shown.

Fig. 4.

PrdH-mediated cleavage requires PrdB and C421 to install the pyruvoyl moiety in PrdA, allowing the production of catalytically active Prd fragments. A) SDS-PAGE showing the requirement of a preformed PrdA/B complex and of PrdA_C421 for processing. Different mixtures of PrdA_149-626 or its C421 mutants, PrdB_U151C, and PrdH have been analyzed. B) Detection of the pyruvoyl cofactor in α-PrdA via fluorescence after incubation with fluorescein thiosemicarbazide. C) Recombinant expression of selenocysteine-containing PrdB leads to the same PrdH-mediated PrdA processing as with PrdB_U151C. Note that this experiment was performed with PrdA_164-626, leading to a β-PrdA fragment of nearly identical molecular weight as PrdB (28.6 kDa vs. 27.9 kDa), which explains why these proteins are not separated on the SDS-PAGE. D) Detection of 5-aminovalerate via fluorescence after reaction with o-phthalaldehyde demonstrates the proline reductase activity of PrdA_164-626/PrdB after processing with PrdH.

Fig. 5.

Identification of important motifs in PrdH. A) Sequence conservation within a central stretch of PrdH, based on the alignment of 198 sequences (created with WebLogo (26)). B) Sequence conservation mapped onto the AlphaFold2-model of PrdH (created with ConSurf (27)). Note the high conservation of the exposed residue R32. C) SDS-PAGE analysis shows the requirement of R32 for PrdH-mediated processing of PrdA.

Fig. 6.

PrdA, PrdB, and PrdH engage in different complexes during pyruvoyl formation. SEC profiles of recombinant proteins mixed directly before analysis were recorded to identify complexes that form during the maturation of Prd. From left to right: PrdH co-eluted with PrdA_149-626, indicating interaction between these two proteins in the absence of PrdB. Co-elution of PrdA_149-626, PrdB_U151C and PrdH suggests interaction between all three components, with the processing reaction occurring during the chromatography run. While C421S-substitution in PrdA_149-626 abolishes the processing reaction, co-elution suggests that the interaction with PrdB_U151C and PrdH still prevails. Mixing PrdA_149-626, PrdB_U151C, and PrdH_R32A shows that the PrdH-mutant does not interact with the complex unprocessed PrdA and PrdB at all, pointing at the critical role of R32. Note that monomeric PrdB and the homodimeric PrdH have similar molecular weight, leading to co-elution without complex formation.

Fig. 7.

Coexpression of PrdA, PrdB, and PrdH in E. coli leads to Prd with similar properties as Prd isolated from C. difficile. A) Prd purified from C. difficile 630Δerm (see also Fig. S7). B) Prd generated by coexpression of PrdA, PrdB_U151, and PrdH in E. coli. Small amounts of π-PrdA and PrdH were copurified with Prd, indicating incomplete processing. Note that different molecular weight markers and SDS-PAGE were used in (A) and (B), leading to slightly different apparent molecular weights. C) Comparison of SEC-MALS profiles of natively and recombinantly expressed Prd reveals similar molecular masses.

Fig. 8.

The ΔprdH variant of C. difficile is insensitive to the L-proline concentration in growth media. Both C. difficile 630 (WT) and C. difficile (ΔprdH) were grown anaerobically in CDMM medium under anaerobic conditions supplemented with different concentrations of L-proline (+ L-Pro: 2 g/L; ++ L-Pro: 8 g/L). Data points represent the means of quadruplicate cultures with error bars representing standard deviations.