Curation Notes for cadA2 (VPARA_44980, A0A0H2LVW9) in Variovorax paradoxus TBEA6

1. Gene & Protein Overview

• UniProtKB Accession: A0A0H2LVW9 (A0A0H2LVW9_VARPD, unreviewed, 759 aa, 78,618 Da)
• Gene Symbol / Locus Tag: cadA2 / VPARA_44980 (EMBL:KLN54354.1 on genomic contig JZWI01000024, RefSeq:WP_047786071.1)
• Organism: Variovorax paradoxus strain TBEA6 (NCBITaxon:34073)
• Genomic Context: Encoded on genomic contig JZWI01000024 within the same heavy-metal resistance island as czcA3-czcB-czcC-czcI-czcR-cusS (VPARA_44890-VPARA_44940, KLN54345.1-KLN54350.1), 4 ORFs downstream of cusS (VPARA_44980, KLN54354.1).
• Domain Architecture & Key Sequence Motifs (Verified against SQ block):
• N-terminal Heavy-Metal-Associated (HMA) cytoplasmic domain (CDD:cd00371, InterPro:IPR006121, SUPFAM:SSF55008, residues ~1..120): Contains a cysteine-rich metal-binding motif 26-CCGSTAC-32 (Cys26, Cys27, Cys32) and 57-MDC-59 (Cys59).
• Transmembrane domain M (InterPro:IPR023298): UniProt RuleBase:RU362081 annotates six transmembrane helices at 140..160, 166..186, 198..214, 368..390, 705..726, and 732..750, while residues 402..421 (402-KALVLLVIACPCALVISTPV-421) form the canonical $\text{P}_{1\text{B}}$-type intramembrane metal-translocation helix carrying the invariant Cys410-Pro411-Cys412 (410-CPC-412) motif.
• Cytoplasmic Actuator (A) domain (Pfam:PF00122, InterPro:IPR059000, FT DOMAIN 251..352): Contains the conserved 296-TGES-299 phosphatase motif.
• Cytoplasmic HAD-like phosphorylation (P) and nucleotide-binding (N) domains (Pfam:PF00702, InterPro:IPR023299, InterPro:IPR018303, PROSITE:PS00154): Contains the invariant catalytic phosphorylation aspartate Asp454 within the 454-DKTGTIT-460 P-type ATPase motif (PROSITE:PS00154) and the conserved $\text{Mg}^{2+}$-coordinating hinge motif 650-GDGINDAPALA-660.
• Family & Subfamily Classification: NCBIfam:TIGR01512 (ATPase-IB2_Cd, $\text{Cd}^{2+}$/$\text{Zn}^{2+}$-exporting $\text{P}_{1\text{B}-2}$-type ATPase), NCBIfam:TIGR01525 (ATPase-IB_hvy), InterPro:IPR027256 (P-typ_ATPase_IB), PANTHER:PTHR48085:SF5 (CADMIUM_ZINC-TRANSPORTING ATPASE HMA4-RELATED), EC 7.2.2.12 (RHEA:20621, P-type $\text{Zn}^{2+}$ transporter).

2. Literature & Comparative Evidence

• PMID:9405611(https://pubmed.ncbi.nlm.nih.gov/9405611/) (Rensing, Mitra & Rosen 1997, Proc Natl Acad Sci U S A 94:14326-14331): Identified E. coli ZntA as the prototype bacterial $\text{Zn}^{2+}$/$\text{Cd}^{2+}$-translocating $\text{P}_{1\text{B}}$-type ATPase, showing that everted membrane vesicles catalyze vanadate-sensitive, ATP-dependent accumulation of $^{65}\text{Zn(II)}$ and $^{109}\text{Cd(II)}$ ("Everted membrane vesicles from a wild-type strain accumulated 65Zn(II) and 109Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases.").
• PMID:12867443(https://pubmed.ncbi.nlm.nih.gov/12867443/) (Legatzki et al. 2003, J Bacteriol 185:4354-4361): Demonstrated the physiological interplay between the CzcCBA RND efflux complex and the two soft-metal-transporting $\text{P}_{1\text{B}}$-type ATPases CadA and ZntA in Ralstonia metallidurans (Cupriavidus metallidurans CH34), establishing that both CadA and ZntA export cytoplasmic $\text{Cd}^{2+}$ and $\text{Zn}^{2+}$ across the inner membrane ("Cadmium and zinc are removed from cells of Ralstonia metallidurans by the CzcCBA efflux pump and by two soft-metal-transporting P-type ATPases, CadA and ZntA.").

3. Review of Existing GOA Annotations (16 IEA rows)

1. GO:0000166 (nucleotide binding, enables, IEA GO_REF:0000002 from InterPro:IPR023299) -> TOO_GENERAL:
2. True but uninformative parent term already entailed by the accepted child term GO:0005524 (ATP binding).
3. GO:0005524 (ATP binding, enables, IEA GO_REF:0000120 from InterPro:IPR001757, UniRule:UR000169778) -> ACCEPT:
4. Core cosubstrate-binding activity of the cytoplasmic N/P domains (InterPro:IPR023299, InterPro:IPR001757), required for formation of the Asp454 acyl-phosphate intermediate (454-DKTGTIT-460).
5. GO:0005886 (plasma membrane, located_in, IEA GO_REF:0000120 from UniProtKB-SubCell:SL-0039, UniRule:UR000169778) -> ACCEPT:
6. Core subcellular localization of the multi-pass inner-membrane $\text{P}_{1\text{B}}$-type efflux ATPase (RuleBase:RU362081).
7. GO:0006812 (monoatomic cation transport, involved_in, IEA GO_REF:0000002 from InterPro:IPR027256) -> TOO_GENERAL:
8. True but uninformative parent process already entailed by the accepted specific child terms GO:0071577 (zinc ion transmembrane transport) and GO:0070574 (cadmium ion transmembrane transport).
9. GO:0015086 (cadmium ion transmembrane transporter activity, enables, IEA GO_REF:0000118 from PANTHER:PTN002458237) -> ACCEPT:
10. Core molecular function of CadA/ZntA-family $\text{P}_{1\text{B}-2}$-type ATPases (NCBIfam:TIGR01512 ATPase-IB2_Cd, PANTHER:PTHR48085:SF5), which export both $\text{Cd}^{2+}$ and $\text{Zn}^{2+}$ across the cytoplasmic membrane (PMID:9405611, PMID:12867443).
11. GO:0016020 (membrane, located_in, IEA GO_REF:0000120 from InterPro:IPR001757, InterPro:IPR027256, UniProtKB-SubCell:SL-0162, PANTHER:PTN002458237) -> TOO_GENERAL:
12. True but uninformative parent cellular component already entailed by GO:0005886 (plasma membrane).
13. GO:0016463 (P-type zinc transporter activity, enables, IEA GO_REF:0000120 from RHEA:20621, EC:7.2.2.12) -> ACCEPT:
14. Primary catalytic and transport activity (EC 7.2.2.12, RHEA:20621) coupling ATP hydrolysis at Asp454 (454-DKTGTIT-460) to transmembrane $\text{Zn}^{2+}$ export via the intramembrane 410-CPC-412 site (PMID:9405611, PMID:12867443).
15. GO:0016887 (ATP hydrolysis activity, enables, IEA GO_REF:0000002 from InterPro:IPR001757) -> TOO_GENERAL:
16. True but uninformative parent molecular function already entailed by GO:0016463 (P-type zinc transporter activity, which is an is_a descendant of GO:0019829 $\rightarrow$ GO:0042626 $\rightarrow$ GO:0016887).
17. GO:0019829 (ATPase-coupled monoatomic cation transmembrane transporter activity, enables, IEA GO_REF:0000002 from InterPro:IPR027256) -> TOO_GENERAL:
18. True but uninformative parent molecular function already entailed by the accepted specific child term GO:0016463 (P-type zinc transporter activity).
19. GO:0022857 (transmembrane transporter activity, enables, IEA GO_REF:0000118 from PANTHER:PTN002458237) -> TOO_GENERAL:
    • True but uninformative parent molecular function already entailed by GO:0016463 and GO:0015086.
20. GO:0030001 (metal ion transport, involved_in, IEA GO_REF:0000120 from ARBA:ARBA00028072, PANTHER:PTN002458237) -> TOO_GENERAL:
    • True but uninformative parent process already entailed by GO:0071577 (zinc ion transmembrane transport) and GO:0070574 (cadmium ion transmembrane transport).
21. GO:0046872 (metal ion binding, enables, IEA GO_REF:0000002 from InterPro:IPR006121, InterPro:IPR036163) -> MODIFY to GO:0008270 (zinc ion binding) and GO:0046870 (cadmium ion binding):
    • Supported by the N-terminal heavy-metal-associated (HMA) regulatory domain (CDD:cd00371, InterPro:IPR006121, SUPFAM:SSF55008, containing 26-CCGSTAC-32) and the intramembrane 410-CPC-412 metal-coordination site of this $\text{P}_{1\text{B}-2}$-subfamily $\text{Zn}^{2+}$/$\text{Cd}^{2+}$ ATPase (NCBIfam:TIGR01512, PANTHER:PTHR48085:SF5; PMID:9405611, PMID:12867443); refined to the specific descendants GO:0008270 (zinc ion binding) and GO:0046870 (cadmium ion binding).
22. GO:0055085 (transmembrane transport, involved_in, IEA GO_REF:0000118 from PANTHER:PTN002458237) -> TOO_GENERAL:
    • True but uninformative parent process already entailed by GO:0071577 and GO:0070574.
23. GO:0070574 (cadmium ion transmembrane transport, involved_in, IEA GO_REF:0000108 from GO:0015086) -> ACCEPT:
    • Core biological process mediated by CadA2 (NCBIfam:TIGR01512, PANTHER:PTHR48085:SF5; PMID:9405611, PMID:12867443).
24. GO:0071577 (zinc ion transmembrane transport, involved_in, IEA GO_REF:0000108 from GO:0016463) -> ACCEPT:
    • Core biological process mediated by CadA2 (EC 7.2.2.12, RHEA:20621; PMID:9405611, PMID:12867443).
25. GO:0098655 (monoatomic cation transmembrane transport, involved_in, IEA GO_REF:0000108 from GO:0019829) -> TOO_GENERAL:
    • True but uninformative parent process already entailed by GO:0071577 and GO:0070574.