A Single DNA Point Mutation Leads to the Formation of a Cysteine–Tyrosine Crosslink in the Cysteine Dioxygenase from Bacillus subtilis

Abstract

Cysteine dioxygenase (CDO) is a non-heme ironcontaining enzyme that catalyzes the oxidation of cysteine (Cys) to cysteine sulfinic acid (CSA). Crystal structures of eukaryotic CDOs revealed the presence of an unusual crosslink between the sulfur of a cysteine residue (C93 in Mus musculus CDO, MmCDO) and a carbon atom adjacent to the phenyl group of a tyrosine residue (Y157). Formation of this crosslink occurs over time as a byproduct of catalysis and increases the catalytic efficiency of CDO by at least 10-fold. Interestingly, in bacterial CDOs, the residue corresponding to C93 is replaced by a highly conserved glycine (G82 in Bacillus subtilis CDO, BsCDO), which precludes the formation of a C−Y crosslink in these enzymes; yet bacterial CDOs achieve turnover rates paralleling those of fully crosslinked eukaryotic CDOs. In the present study, we prepared the G82C variant of BsCDO to determine if a single DNA point mutation could lead to C−Y crosslink formation in this enzyme. We used gel electrophoresis, peptide mass spectrometry, electron paramagnetic resonance spectroscopy, and kinetic assays to characterize this variant alongside the natively crosslinked wild-type (WT) MmCDO and the natively noncrosslinked WT BsCDO. Collectively, our results provide compelling evidence that the G82C BsCDO variant is indeed capable of C−Y crosslink formation. Our kinetic studies indicate that G82C BsCDO has a reduced catalytic efficiency compared to WT BsCDO and that activity increases as the ratio of crosslinked to non-crosslinked enzyme increases. Finally, by carrying out a bioinformatic analysis of the CDO family, we were able to identify a large number of putatively crosslinked bacterial CDOs, the majority of which are from Gram-negative pathogenic bacteria.

Kinetic and Spectroscopic Investigation of the Y157F and C93G/Y157F Variants of Cysteine Dioxygenase: Dissecting the Roles of the Second-Sphere Residues C93 and Y157

“…Our results are consistent with, and significantly expand upon, the findings from a recent study of Bacillus subtilis CDO ( Bs CDO), a representative member of bacterial CDOs that are unable to form the Cys–Tyr cross-link due to the conserved replacement of the cysteine by a glycine at position 93 ( Mm CDO numbering) while nevertheless maintaining the analogous tyrosine at position 157. , Despite lacking the Cys–Tyr cross-link, the bacterial CDOs investigated thus far exhibit similar catalytic efficiencies as Mm CDO, emphasizing that the cross-link itself is not necessary for high turnover rates by CDOs that lack a free thiolate near the active site . Interestingly, the G82C variant of Bs CDO, in which the glycine at the position corresponding to C93 in Mm CDO was replaced by a cysteine, was shown to be capable of forming a Cys–Tyr cross-link similar to Mm CDO . The enzymatic efficiency of this variant was found to correlate with the degree of cross-link formation, as observed for Mm CDO.…”