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Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

ABSTRACT: Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.

SUBMITTER: Wenning L

PROVIDER: S-EPMC4725276 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

Wenning Leonie L Stöveken Nadine N Wübbeler Jan Hendrik JH Steinbüchel Alexander A

Applied and environmental microbiology 20151120 3

Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropiona ...[more]

PMID: 26590284

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Project description:The gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha strain H16 is known for its narrow carbohydrate utilization range, which limits its use for biotechnological production of polyhydroxyalkanoates and possibly other products from renewable resources. To broaden its substrate utilization range, which is for carbohydrates and related compounds limited to fructose, N-acetylglucosamine, and gluconate, strain H16 was engineered to use mannose and glucose as sole carbon sources for growth. The genes for a facilitated diffusion protein (glf) from Zymomonas mobilis and for a glucokinase (glk), mannofructokinase (mak), and phosphomannose isomerase (pmi) from Escherichia coli were alone or in combination constitutively expressed in R. eutropha strain H16 under the control of the neokanamycin or lac promoter, respectively, using an episomal broad-host-range vector. Recombinant strains harboring pBBR1MCS-3::glf::mak::pmi or pBBR1MCS-3::glf::pmi grew on mannose, whereas pBBR1MCS-3::glf::mak and pBBR1MCS-3::glf did not confer the ability to utilize mannose as a carbon source to R. eutropha. The recombinant strain harboring pBBR1MCS-3::glf::pmi exhibited slower growth on mannose than the recombinant strain harboring pBBR1MCS-3::glf::mak::pmi. These data indicated that phosphomannose isomerase is required to convert mannose-6-phosphate into fructose-6-phosphate for subsequent catabolism via the Entner-Doudoroff pathway. In addition, all plasmids also conferred to R. eutropha the ability to grow in the presence of glucose. The best growth was observed with a recombinant R. eutropha strain harboring plasmid pBBR1MCS-2::P(nk)::glk::glf. In addition, expression of the respective enzymes was demonstrated at the transcriptional and protein levels and by measuring the activities of mannofructokinase (0.622 ± 0.063 U mg(-1)), phosphomannose isomerase (0.251 ± 0.017 U mg(-1)), and glucokinase (0.518 ± 0.040 U mg(-1)). Cells of recombinant strains of R. eutropha synthesized poly(3-hydroxybutyrate) to ca. 65 to 67% (wt/wt) of the cell dry mass in the presence of 1% (wt/vol) glucose or mannose as the sole carbon sources.

Dataset Information

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

Publications

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

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