DusA is the tRNA-dihydrouridine(20/20a) synthase of Sinorhizobium meliloti, an FMN-dependent flavoenzyme of the Dus (COG0042) family that reduces the C5-C6 double bond of uridines at positions 20 and 20a in the tRNA D-loop, using NADPH or NADH as the electron source. It belongs to the DusA subfamily (HAMAP MF_02041), whose members bind the tRNA elbow in an orientation set by subfamily-specific binding-signature residues and so modify U20/U20a rather than the U16 and U17 sites of DusC and DusB. The protein keeps the catalytic cysteine (Cys83 in the UniProt numbering, equivalent to E. coli DusA Cys114), the FMN-binding residues and the subfamily signature residues, and carries two short insertions in its C-terminal helical domain. The UniProt sequence starts within the conserved N-terminal FMN-binding segment, and the open reading frame continues upstream, so the native protein is probably longer at its N-terminus. No rhizobial DusA has been studied experimentally; the function rests on the E. coli and Thermus thermophilus enzymes, where D20/D20a formation tunes tRNA structure and the decoding of specific codons.
The protein sequence of this entry may not correspond to a single real gene product (for example, two neighbouring genes joined into one model, or a truncated model). Read the name, features and annotations of the entry with this in mind.
WRONG START SITE SUSPECTED ORTHOLOG COMPARISONGENOMIC SEQUENCE
The 359-residue UniProt/EMBL model (CAC46484) starts at MIDWTD, which aligns to E. coli DusA Met33, so the first residue (Pro32) of the HAMAP MF_02041 FMN-binding feature at E. coli 32-34 is missing; its codon lies just upstream in an open frame. The feature therefore cannot be placed on Q92P85, which is why UniProt flags missing conserved residues. The current RefSeq model for the same locus (SM_RS09780, WP_015241224.1) starts 26 codons upstream and contains the motif, as does the 398-residue KBase model from strain USDA1810, whose different N-terminal 39 residues probably come from a dusA-associated integrase island. The start used in vivo is not known. Functional statements apply to the full-length protein, and the domain and site analysis is unaffected beyond that one residue.
Supported by:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000049 tRNA binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: True but peripheral: DusA must bind its tRNA substrate, and the E. coli enzyme binds tRNA directly, but binding is part of the catalytic activity rather than a separate function. Reason: E. coli DusA binds tRNA in a two-step mechanism and prefers modified tRNA (PMID:42080256), and the tRNA-contact and subfamily-signature residues are conserved here (dusA-bioinformatics/RESULTS.md). The UniRule mapping is sound. The term is as precise as the binding evidence allows, but it describes substrate binding by an enzyme whose core function is the synthase activity, so it is kept as non-core. Supporting Evidence: PMID:42080256 We find that DusA binds tRNA via a two-step mechanism involving a local structural rearrangement and exhibits a higher affinity for previously modified tRNA compared to unmodified tRNA. file:RHIME/dusA/dusA-bioinformatics/RESULTS.md tRNA-interacting E. coli N111 and R201 are conserved (N80, R169) |
| GO:0002943 tRNA dihydrouridine synthesis | IEA GO_REF:0000120 | ACCEPT | Summary: Correct core process. DusA catalyses the conversion of U20 and U20a to dihydrouridine, and this is the most specific process term available. Reason: Q92P85 is a DusA-subfamily member (HAMAP MF_02041, InterPro IPR004653), with the catalytic cysteine and subfamily signature residues conserved. E. coli DusA is the U20/U20a enzyme in vivo (PMID:29294097, PMID:39460624). The process is directly performed by the enzyme. Supporting Evidence: file:RHIME/dusA/dusA-uniprot.txt SIMILARITY: Belongs to the Dus family. DusA subfamily. PMID:29294097 DusC and DusB are mono-site specific flavoenzymes involved in D16 and D17 biosynthesis, respectively, while DusA is a U20/U20a bi-site specific enzyme file:RHIME/dusA/dusA-bioinformatics/RESULTS.md E. coli C114 -> Q92P85 C83 (KBase 122) conserved E.coli NLNVGCPSDRV Q92P85 NMNVGCPSDRV |
| GO:0008033 tRNA processing | IEA GO_REF:0000002 | MODIFY | Summary: Correct but general; the gene already carries the specific descendant GO:0002943 tRNA dihydrouridine synthesis. Reason: Dihydrouridine formation is a tRNA modification, and GO:0002943 is a descendant of GO:0008033 that the gene already carries (IEA, UniRule and InterPro IPR004653). The Dus conserved-site signature behind this row supports the specific process equally well. Proposed replacements: tRNA dihydrouridine synthesis Supporting Evidence: PMID:11983710 The results showed that the COG0042 gene family is responsible for tRNA-dihydrouridine synthase activity in E. coli. |
| GO:0010181 FMN binding | IEA GO_REF:0000104 | ACCEPT | Summary: Correct. Dus enzymes carry FMN as the prosthetic group that is reduced by NAD(P)H and transfers a hydride to the uridine, and the FMN-binding residues are conserved here. Reason: FMN is the catalytic cofactor of the Dus family (PMID:22123979), and in the Thermus Dus-tRNA complex U20 stacks on the FMN isoalloxazine ring. All HAMAP MF_02041 FMN-binding residues are conserved in Q92P85 except E. coli Pro32, which the truncated gene model omits, and Leu34, which is conservatively replaced by Ile (dusA-bioinformatics/RESULTS.md). Cofactor binding is integral to the core catalytic function. Supporting Evidence: PMID:22123979 Dihydrouridine synthase (Dus) catalyzes the D formation of tRNA through reduction of uracil base with flavin mononucleotide (FMN) as a cofactor. file:RHIME/dusA/dusA-uniprot.txt Name=FMN; Xref=ChEBI:CHEBI:58210; |
| GO:0017150 tRNA dihydrouridine synthase activity | IEA GO_REF:0000120 | MODIFY | Summary: Correct but general; the gene already carries the position-specific descendants for U20 and U20a. Reason: DusA-subfamily enzymes are site-specific for U20 and U20a (PMID:29294097), and the specificity follows from subfamily binding signatures (PMID:25902496) that are conserved here. GO:0102264 and GO:0102266 are both is_a descendants of GO:0017150 and both are already in GOA for this protein, so the generic activity is refined to them. Proposed replacements: tRNA-dihydrouridine20 synthase activity tRNA-dihydrouridine20a synthase activity Supporting Evidence: PMID:29294097 DusC and DusB are mono-site specific flavoenzymes involved in D16 and D17 biosynthesis, respectively, while DusA is a U20/U20a bi-site specific enzyme PMID:25902496 The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids |
| GO:0050660 flavin adenine dinucleotide binding | IEA GO_REF:0000002 | REMOVE | Summary: Wrong cofactor. Dus enzymes are FMN flavoenzymes; FAD binding comes from a broad InterPro2GO mapping of the Dus conserved-site signature. Reason: The flavin of the Dus family is FMN, shown in the Thermus Dus-tRNA crystal structures (PMID:22123979), and UniProt and HAMAP record FMN as the cofactor of this protein. No Dus enzyme is known to bind FAD. The row is an electronic mapping from IPR018517 (tRNA-dihydrouridine synthase conserved site) that also puts FAD binding on E. coli DusA (QuickGO, 2026-10-30), next to the correct FMN binding row; the upstream defect is recorded below. Supporting Evidence: PMID:22123979 In the active site, U20 is stacked on the isoalloxazine ring of FMN file:RHIME/dusA/dusA-uniprot.txt Name=FMN; Xref=ChEBI:CHEBI:58210; |
| GO:0102264 tRNA-dihydrouridine20 synthase activity | IEA GO_REF:0000003 | ACCEPT | Summary: Correct core activity. EC 1.3.1.91 is the U20 reaction of the DusA subfamily, to which this protein belongs. Reason: The EC assignment comes from HAMAP MF_02041. DusA reduces U20 in E. coli tRNAs (PMID:29294097), and this protein keeps the catalytic cysteine and the subfamily tRNA-binding signature (dusA-bioinformatics/RESULTS.md). Supporting Evidence: file:RHIME/dusA/dusA-uniprot.txt DR HAMAP; MF_02041; DusA_subfam; 1. PMID:29294097 DusC and DusB are mono-site specific flavoenzymes involved in D16 and D17 biosynthesis, respectively, while DusA is a U20/U20a bi-site specific enzyme |
| GO:0102266 tRNA-dihydrouridine20a synthase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Correct core activity. DusA-subfamily enzymes modify U20a as well as U20. Reason: E. coli DusA is bi-site specific for U20 and U20a (PMID:29294097), and the Rhea reactions behind this row come from the same HAMAP rule. Not every tRNA has a U20a, so this activity applies to the subset of tRNAs with a uridine at that position. Supporting Evidence: PMID:29294097 DusC and DusB are mono-site specific flavoenzymes involved in D16 and D17 biosynthesis, respectively, while DusA is a U20/U20a bi-site specific enzyme |
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Download this section (compressed HTML)Q: Which start codon does S. meliloti use for dusA? The RefSeq model (WP_015241224.1) adds 26 residues that include the equivalent of E. coli DusA FMN-binding Pro32, which is missing from Q92P85.
Q: Do strains with a dusA-associated integrase island, such as USDA1810, make a functional DusA from the island-supplied 5' end?
Q: Why does the S. meliloti dusA transposon mutant grow better in 10 mM perchlorate (Fitness Browser data, as shown on the KBase dashboard), and is this linked to NADPH-dependent redox sensitivity of Dus activity?
Experiment: Map dihydrouridines in tRNAs of S. meliloti wild type and a dusA deletion (AlkAnilineSeq or LC-MS of tRNA digests) to confirm loss of D20/D20a.
Type: tRNA modification mapping
Experiment: Determine the N-terminus of native DusA by N-terminal proteomics or ribosome profiling with translation-initiation inhibitors in strains 1021 and USDA1810.
Type: gene model validation
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