Mercaptosuccinate metabolism in Variovorax paradoxus strain B4—a proteomic approach

Abstract

Variovorax paradoxus B4 was isolated due to its ability to degrade the organic thiol compound mercaptosuccinate, which could be a promising precursor for novel polythioesters. The analysis of the proteome of this Gram-negative bacterium revealed several proteins with significantly increased expression during growth of cells with mercaptosuccinate as carbon source when compared to cells grown with gluconate or succinate. Among those, a large number of proteins involved in amino acid metabolism were identified, e.g., adenosylhomocysteinase and glutamate-ammonia ligase. Additionally, detection of superoxide dismutase strengthened the assumption of enhanced stress levels in mercaptosuccinate-grown cells. Several isoforms of a rhodanese domain-containing protein exhibited particularly increased expression during growth with mercaptosuccinate in comparison to gluconate (factor 14.2, stationary phase) or to succinate (factor 15.4, stationary phase). Besides this, augmented expression of the hypothetical protein VAPA_1c41240 raised attention. VAPA_1c41240 exhibited up to 13.3-fold (mercaptosuccinate vs gluconate) or 9.5-fold (mercaptosuccinate vs succinate) increased expression levels, and in silico searches revealed that this protein might be a thiol dioxygenase. Based on these results, a novel degradation pathway is proposed for mercaptosuccinate. The newly identified putative mercaptosuccinate dioxygenase could convert mercaptosuccinate to sulfinosuccinate by the introduction of two molecules of oxygen. Subsequently, sulfinosuccinate would be cleaved into succinate and sulfite either by a yet unknown enzyme, by spontaneous hydrolysis, or by the putative mercaptosuccinate dioxygenase itself. Succinate could then enter the central metabolism, while detoxification of sulfite could be achieved by the previously identified putative molybdopterin oxidoreductase. Biochemical studies will be done in the future to confirm this pathway.

Biochemical characterization and essentiality of fumarate hydratase

“…This common observation of higher IC 50 values observed for both cell types could be due to the low intracellular availability of the drug (owing to the hydrophilic nature of the molecule and hence poor transport) and/or due to metabolism leading to degradation of MSA. MSA dioxygenase, an enzyme that converts mercaptosuccinic acid to succinate is present in the bacterium Variovorax paradoxus (48,49). We, however, could not find homologues of the enzyme in E. coli or P. falciparum.…”

Mercaptosuccinate Dioxygenase, a Cysteine Dioxygenase Homologue, from Variovorax paradoxus Strain B4 Is the Key Enzyme of Mercaptosuccinate Degradation

“…Identification of the Putative MS Dioxygenase-In the course of proteomic studies with V. paradoxus strain B4 (17), several isoforms of a protein with significant up-regulation during growth with MS as carbon source in comparison to growth with gluconate or succinate were identified. This protein was annotated as a hypothetical protein (VAPA_1c41240); however, it showed 46% amino acid similarity to an annotated cysteine dioxygenase of Ralstonia pickettii DTP0602 (17).…”

“…Only recently, another putative novel thiol dioxygenase was identified during proteomic studies with V. paradoxus B4 indicating that this protein might be a mercaptosuccinate dioxygenase and would therefore represent the key enzyme in the degradation of MS in this bacterium (17). Although the putative thiol dioxygenase was originally annotated as a hypothetical protein, further in silico analyses resulted in a hit for the COG5553 domain in the NCBI database comprising metal-dependent enzymes of the double-stranded ␤ helix superfamily (17).…”

“…Although the putative thiol dioxygenase was originally annotated as a hypothetical protein, further in silico analyses resulted in a hit for the COG5553 domain in the NCBI database comprising metal-dependent enzymes of the double-stranded ␤ helix superfamily (17). Additionally, InterProScan 5 for functional analysis of proteins (EMBL-EBI, Hinxton, United Kingdom) revealed an RmlC-like cupin domain and an RmlC-like jellyroll-fold, representing the aforementioned conserved ␤-barrel core of thiol dioxygenases (17). These findings supported the assumption that the hypothetical protein might actually be a thiol dioxygenase.…”

“…These findings supported the assumption that the hypothetical protein might actually be a thiol dioxygenase. In V. paradoxus B4, MS is supposedly converted to sulfinosuccinate by the aforementioned putative MS dioxygenase and then cleaved into succinate and sulfite either by a so far unknown enzyme, by spontaneous hydrolysis, or even by the putative MS dioxygenase itself (17). To verify the postulated reaction of the putative MS dioxygenase and to further unravel the degradation of MS, the enzyme was heterologously expressed and characterized in this study.…”