| Section | Residue Position (MCR-1) / Comparison Item | Equivalent in NmEptA | Role/Function | Conservation Status | Evidence of Essentiality | Notes |
|---|---|---|---|---|---|---|
| Active site | Thr285 | Thr280 | Catalytic nucleophile; forms covalent pEtN-enzyme intermediate and coordinates catalytic Zn2+ | Identical in MCR-1 and NmEptA; absolutely conserved catalytic Thr among Class I lipid A PETs | T285A abolishes catalytic activity; deep mutational scanning found stringent WT preference at this position; phosphorylated Thr observed structurally (pqac-00000017, pqac-00000018, pqac-00000019, pqac-00000022, pqac-00000023) | Decisive evidence against pseudo-enzyme interpretation |
| Active site | Glu246 | Glu240 | Zn2+ coordination; proton shuttle in first catalytic step | Identical between MCR-1 and NmEptA (pqac-00000017, pqac-00000018) | Conservative substitution E246D significantly decreases polymyxin resistance/activity; strong WT enrichment in mutational scan (pqac-00000020, pqac-00000002) | Core catalytic residue supporting intact chemistry |
| Active site | Asp465 | Asp452 | Zn2+ coordination; active-site metal binding | Equivalent catalytic Asp conserved in NmEptA and Class I PETs (pqac-00000017, pqac-00000022) | D465E reduces steady-state protein and activity/resistance; WT residue strongly preferred (pqac-00000019, pqac-00000020) | Supports preserved metalloenzyme architecture |
| Active site | His466 | His453 | Zn2+ coordination; catalytic metal environment/productive active-site geometry | Identical catalytic His in MCR-1 and NmEptA (pqac-00000017, pqac-00000022) | Point-mutation evidence and Class I PET conservation indicate essentiality; highlighted in mechanistic models (pqac-00000003, pqac-00000021, pqac-00000022) | Often discussed with Asp465 as part of catalytic metal-binding set |
| Active site | His395 | His383 | Second Zn2+ coordination / catalytic support in di-zinc model | Conserved among Class I PETs (pqac-00000018, pqac-00000022) | Mechanistic/structural evidence supports importance; included among key charged/polar pocket residues (pqac-00000003, pqac-00000021) | Second Zn2+ occupancy may vary by structure/preparation, but residue is conserved |
| Active site | Thr247 | Not explicitly mapped in retrieved context | Hydrogen-bond network adjacent to catalytic center | Strong WT preference in MCR-1 mutational scan (pqac-00000020) | Mutations significantly decrease activity/resistance in active-site randomization study (pqac-00000002, pqac-00000020) | Supports intact substrate-positioning network |
| Active site | Leu120 | Not explicitly mapped in retrieved context | Hydrophobic interaction in substrate tunnel / PE accommodation | Strong WT preference in mutational scan; part of tunnel-shaped cavity (pqac-00000000, pqac-00000020) | Identified as essential in deep mutational scanning (pqac-00000002, pqac-00000020) | Important for donor-substrate handling rather than direct metal ligation |
| Active site | Phe93 | Not explicitly mapped in retrieved context | Aromatic residue at substrate tunnel entrance | Present in MCR-1 active-site tunnel architecture (pqac-00000000) | Included among tested active-site/tunnel residues affecting function in mutagenesis framework (pqac-00000000, pqac-00000004) | Contributes to PE headgroup/acyl-chain access path |
| Active site | Tyr97 | Not explicitly mapped in retrieved context | Aromatic residue at tunnel entrance | Present in active-site entrance architecture (pqac-00000000) | Functional importance inferred from active-site residue survey and tunnel design (pqac-00000000, pqac-00000004) | Tunnel-shaping residue |
| Active site | Met105 | Not explicitly mapped in retrieved context | Hydrophobic tunnel/entry residue | Present in donor-substrate tunnel (pqac-00000000) | Functional contribution inferred from active-site mutational framework (pqac-00000000, pqac-00000004) | Supports PE acyl-chain accommodation |
| Active site | Asn108 | Not explicitly mapped in retrieved context | Polar residue in tunnel body/substrate recognition | Present in tunnel body (pqac-00000000) | Functional contribution inferred from active-site mapping (pqac-00000000, pqac-00000004) | Likely participates in donor positioning |
| Active site | Ala109 | Not explicitly mapped in retrieved context | Hydrophobic tunnel residue | Present in tunnel architecture (pqac-00000000) | Functional contribution inferred from active-site mapping (pqac-00000000, pqac-00000004) | Donor-channel shaping residue |
| Active site | Glu116 | Not explicitly mapped in retrieved context | Polar/charged pocket residue near active site | Included in predicted active-site pocket of full-length structure (pqac-00000021) | Mechanistic importance supported by structural placement near catalytic center (pqac-00000021) | May help orient donor/acceptor phosphates |
| Active site | Thr117 | Not explicitly mapped in retrieved context | Hydrophilic residue at tunnel entrance | Present in tunnel entrance architecture (pqac-00000000) | Functional contribution inferred from active-site mapping (pqac-00000000, pqac-00000004) | Likely contributes to PE headgroup recognition |
| Active site | Lys333 | Not explicitly mapped in retrieved context | Polar/charged pocket residue in active-site environment | Included in predicted active-site pocket (pqac-00000021) | Mechanistic importance inferred from full-length structural model (pqac-00000021) | Candidate role in phosphate-group stabilization |
| Active site | Leu477 | Not explicitly mapped in retrieved context | Hydrophobic residue in tunnel body | Identified in tunnel-shaped cavity (pqac-00000000) | Functional contribution inferred from active-site mapping (pqac-00000000) | Likely contributes to donor acyl-chain fit |
| Placement | MCR-1 vs NmEptA (nearest characterized homolog) | NmEptA full-length lipid A PET | Same enzyme family/subfamily: Class I lipid A phosphoethanolamine transferases | MCR-1 shares ~36% overall sequence identity with NmEptA; catalytic domain more conserved than TM domain; core catalytic residues identical (pqac-00000012, pqac-00000017, pqac-00000010) | Strong structural/evolutionary support for orthologous biochemical function (pqac-00000010, pqac-00000011, pqac-00000014) | Closest characterized homolog for function inference is NmEptA, not a generic phosphatase |
| Placement | Superfamily assignment | Alkaline phosphatase superfamily | Fold-level classification | Shared by MCR-1, EptA/NmEptA, and other PETs such as BcsG (pqac-00000010, pqac-00000011, pqac-00000014, pqac-00000024) | Structural evidence definitive (pqac-00000010, pqac-00000011, pqac-00000024) | Explains TreeGrafter susceptibility to over-broad family-level transfer |
| Placement | Donor substrate specificity | PE donor also used by EptA/NmEptA | MCR-1 transfers pEtN from phosphatidylethanolamine, not free phosphate | Conserved within Class I lipid A PETs (pqac-00000000, pqac-00000003, pqac-00000018) | Supported by biochemical and mechanistic studies (pqac-00000000, pqac-00000003) | Seed GO term misses phosphoethanolamine-specific chemistry |
| Placement | Acceptor/site selectivity: MCR-1 | Compared with EptA and other PETs | Lipid A phosphate is acceptor; MCR-1 preferentially modifies 4'-phosphate | 2024 comparative PET study: MCR-1, MCR-3, PET-C are 4'-selective; EptA and MCR-9 prefer 1-phosphate (pqac-00000015) | Experimental comparative phenotype/MS evidence (pqac-00000015) | Key subfamily-level distinction not captured by GO:0016776 |
| Placement | MCR-1 vs EptA | EptA/NmEptA modifies lipid A but often at 1-phosphate | Sibling/near-neighbor activities within same PET family; same broad chemistry, different site selectivity and phenotype | Both are lipid A PETs with common ancestry and conserved catalytic scaffold (pqac-00000012, pqac-00000015, pqac-00000016) | Comparative evidence supports same branch but distinct functional granularity (pqac-00000015, pqac-00000016) | Seed term is too general across these subfamilies |
| Placement | MCR-1 vs other PETs (EptB, CptA/EptC, BcsG) | Distinct PET clades/substrates | Different acceptor substrates despite shared fold: Kdo/core sugars/proteins/cellulose vs lipid A | PET family splits into lipid A-modifying, Kdo-modifying, and inner-core-modifying clades; BcsG modifies cellulose (pqac-00000015, pqac-00000024) | Strong evidence for within-superfamily functional diversity (pqac-00000015, pqac-00000024) | Demonstrates why a superfamily-level annotation is insufficiently specific |
| Placement | GO implication | Broad GO:0016776 vs specific lipid A PET activity | MCR-1 directly fits a phosphoethanolamine transferase/lipid A phosphoethanolamine transferase function more specifically than broad phosphotransferase term | Broad chemistry technically compatible because lipid A phosphate is acceptor, but function is better localized to lipid A PET subfamily (pqac-00000026, pqac-00000027, pqac-00000029, pqac-00000030) | High confidence; limitation is absence in retrieved context of a verified exact GO accession for the specific MF term | Best interpretation: annotation is partially supported but too general |


*Table: This table summarizes residue-level catalytic evidence and subfamily placement for MCR-1. It shows that MCR-1 is an intact, active lipid A phosphoethanolamine transferase in the alkaline phosphatase superfamily and that GO:0016776 is best treated as a broad, too-general annotation.*