DgcA (PP_0310) is the catalytic flavoprotein of the dimethylglycine dehydrogenase system of Pseudomonas putida KT2440. It is a 686-residue soluble protein with an N-terminal NADH:flavin oxidoreductase (Old Yellow Enzyme-type) TIM-barrel domain and a C-terminal FAD/NAD(P)-binding domain, and it is a full-length ortholog (81% identity) of the characterized Chromohalobacter salexigens enzyme DdhC. By that orthology, DgcA oxidatively demethylates N,N-dimethylglycine to sarcosine and formaldehyde, using FAD, and passes the electrons to the adjacent membrane-anchored 2[4Fe-4S] ferredoxin DgcB (PP_0311). DgcB is thought to reduce the electron transfer flavoprotein encoded by the neighbouring PP_0312/PP_0313 genes. This is the dimethylglycine-to-sarcosine step of glycine betaine catabolism, which in Pseudomonas aeruginosa requires the orthologous dgcAB genes. The KT2440 protein has not been purified or assayed directly.
Definition: Catalysis of the reactions: N,N-dimethylglycine + H2O + oxidized 2[4Fe-4S]-[ferredoxin] = sarcosine + formaldehyde + reduced 2[4Fe-4S]-[ferredoxin] + 2 H+, and sarcosine + H2O + oxidized 2[4Fe-4S]-[ferredoxin] = glycine + formaldehyde + reduced 2[4Fe-4S]-[ferredoxin] + 2 H+.
Justification: EC 1.5.7.3 and RHEA:74167/RHEA:74155 describe the DgcA/DdhC reaction, but GO has no corresponding term. The only dimethylglycine and sarcosine dehydrogenase terms (GO:0047865, EC:1.5.8.4; GO:0008480, EC:1.5.8.3) are defined with electron-transfer flavoprotein as the direct acceptor, whereas in the DgcAB system the ETF acts after the DgcB ferredoxin. The closest existing term is the generic parent GO:0033694.
Parent term: oxidoreductase activity, acting on the CH-NH group of donors, iron-sulfur protein as acceptor
Mappings:
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0010181 FMN binding | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: FMN binding is inferred only from the N-terminal Old Yellow Enzyme-type TIM-barrel fold. The flavin identified in the purified 81%-identical ortholog DdhC is noncovalently bound FAD, with no FMN reported. Reason: This InterPro2GO row (GO_REF:0000002) maps IPR001155 (NADH:flavin oxidoreductase/NADH oxidase N-terminal domain) to FMN binding. That is a fold-level inference and the right default for classical Old Yellow Enzymes. The only biochemically characterized member of this subfamily is Chromohalobacter DdhC (Q1QYW1), a full-length ortholog with 81.2% identity. Its purified recombinant form contains noncovalently bound FAD, and its curated cofactors are FAD and NAD(P)+, without FMN. FMN in the TIM barrel cannot be ruled out from the abstract-only source, so the term is not demonstrably wrong. With FAD as the only demonstrated flavin in the subfamily, FMN binding is best treated as a likely over-annotation. The same verdict is given on the ortholog's identical InterPro2GO row in the DdhC review. The supported FAD-binding term is added as a NEW row below. Supporting Evidence: PMID:32631860 existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide file:PSEPK/dgcA/dgcA-bioinformatics/RESULTS.md Q88R24 and Q1QYW1 share 81.2% identity over their full length file:PSEPK/dgcA/dgcA-uniprot.txt DR InterPro; IPR001155; OxRdtase_FMN_N. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: Correct but very general. By orthology to DdhC, DgcA is a CH-NH-oxidizing demethylase whose electron acceptor is the 2[4Fe-4S] ferredoxin DgcB (EC 1.5.7.3). Reason: This InterPro2GO row (GO_REF:0000002, from IPR001155 and IPR023753) gives only the root oxidoreductase term. DgcA is 81.2% identical over its full length to DdhC (Q1QYW1). UniProt curates DdhC reactions as EC 1.5.7.3, demethylating N,N-dimethylglycine (and sarcosine) with an oxidized 2[4Fe-4S] ferredoxin as acceptor, and the ferredoxin is the DgcB ortholog Csal_0991. In P. aeruginosa the orthologous dgcAB genes are required for dimethylglycine-to-sarcosine conversion. The existing GO term for this chemistry is GO:0033694 (oxidoreductase activity, acting on the CH-NH group of donors, iron-sulfur protein as acceptor; EC:1.5.7.-). GO:0047865 dimethylglycine dehydrogenase activity is not used. Its definition ("N,N-dimethylglycine + electron-transfer flavoprotein + H2O = sarcosine + formaldehyde + reduced electron-transfer flavoprotein", EC:1.5.8.4) makes ETF the direct acceptor, but here the ETF sits after DgcB. The EC=1.5.8.- in the TrEMBL record is a submitter annotation (ECO:0000313), not evidence. The replacement is GO:0033694 now, plus the substrate-specific EC 1.5.7.3 child term in proposed_new_terms. Whether the KT2440 enzyme also demethylates sarcosine, as DdhC does in vitro, is untested. Proposed replacements: oxidoreductase activity, acting on the CH-NH group of donors, iron-sulfur protein as acceptor N,N-dimethylglycine/sarcosine dehydrogenase (ferredoxin) activity Supporting Evidence: file:CHRI1/ddhC/ddhC-uniprot.txt Reaction=oxidized 2[4Fe-4S]-[ferredoxin] + N,N-dimethylglycine + H2O = file:CHRI1/ddhC/ddhC-uniprot.txt Regeneration of NAD(P)(+) is achieved by electron transfer to the [4Fe-4S] cluster in the probable membrane-anchored ferredoxin csal_0991 PMID:17951379 strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine file:PSEPK/dgcA/dgcA-bioinformatics/RESULTS.md Q88R24 and Q1QYW1 share 81.2% identity over their full length |
| GO:0050660 flavin adenine dinucleotide binding | ISS PMID:32631860 Role of N,N-Dimethylglycine and Its Catabolism to Sarcosine ... | NEW | Summary: The characterized 81%-identical ortholog DdhC contains noncovalently bound FAD, which is the flavin of the dehydrogenase reaction. Reason: DgcA has the C-terminal FAD/NAD(P)-binding domain (Pfam PF07992, IPR023753) that DdhC uses. Purified DdhC carries noncovalently bound FAD, which in the curated mechanism accepts the hydride from dimethylglycine. GOA has only the fold-based FMN-binding row. FAD binding is transferred by sequence similarity (ISS) from Q1QYW1; FAD has not been measured in the KT2440 protein itself. Supporting Evidence: PMID:32631860 existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide file:PSEPK/dgcA/dgcA-uniprot.txt DR Pfam; PF07992; Pyr_redox_2; 1. file:PSEPK/dgcA/dgcA-bioinformatics/RESULTS.md Q88R24 and Q1QYW1 share 81.2% identity over their full length |
| GO:0031457 glycine betaine catabolic process | ISS PMID:17951379 Identification of two gene clusters and a transcriptional re... | NEW | Summary: DgcA catalyses the dimethylglycine-to-sarcosine step of glycine betaine catabolism, as shown for its orthologs in P. aeruginosa (genetics and 13C tracing) and C. salexigens (enzymology and genetics). Reason: In P. aeruginosa, glycine betaine is degraded by stepwise demethylation: gbcAB turns glycine betaine into dimethylglycine, dgcAB turns dimethylglycine into sarcosine, and soxBDAG turns sarcosine into glycine. 13C NMR tracing showed that dgcAB mutants block the dimethylglycine step. The Chromohalobacter ortholog DdhC catalyses that step directly. DgcA passes the participation test, since it is the enzyme that oxidizes the dimethylglycine. The same ISS-level inference for KT2440 is supported by the conserved dgcA-dgcB-etf locus and 81% identity to DdhC. GO:0031457 is defined as "The chemical reactions and pathways resulting in the breakdown of glycine betaine, N-trimethylglycine". GO has no dimethylglycine catabolic process term. Supporting Evidence: PMID:17951379 strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine PMID:32631860 gene disruption and complementation assays indicated that the csal_0990, csal_0991, csal_0992, and csal_0993 genes are responsible for DMG degradation to sarcosine |
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Download this section (compressed HTML)Q: Which flavin(s) does KT2440 DgcA bind, and does it demethylate sarcosine as well as dimethylglycine?
Q: Does the PP_0312/PP_0313 ETF heterodimer accept electrons directly from reduced DgcB?
Experiment: Purify KT2440 DgcA, determine its bound flavins and nucleotides, and assay dimethylglycine and sarcosine demethylation (formaldehyde release) with NAD(P)+ and with membranes containing DgcB, comparing wild-type and PP_0311-deleted membranes.
Type: biochemical assay
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