Is human AADACL4 a catalytically competent GDXG hydrolase?

Generated by analyze_catalytic_machinery.py. Every sequence, feature and comment below is fetched from the UniProt REST API at run time; nothing is hard-coded from a previous run. Re-running overwrites this file.

Test protein: Q5VUY2 / ADCL4_HUMAN (AADACL4 (human)), 407 aa, UniProt protein existence 3: Inferred from homology.

Part 1 - the catalytic residues, read off the AADACL4 sequence

• Triad residues in sequence order: SDH (Ser/Asp/His expected: True).
• Nucleophile elbow pentapeptide around Ser193: GESVG (canonical GDXG-family form is G-x-S-x-G).
• Residues 119-121: HGG (the GDXG oxyanion-hole motif).
• UniProt MOTIF 119-121: Involved in the stabilization of the negatively charged intermediate by the formation of the oxyanion hole [ECO:0000250]

Part 2 - are those residues in register with characterised relatives?

Each reference is aligned to AADACL4 globally (BLOSUM62, gap open -11, extend -1) and its own annotated catalytic positions are projected onto AADACL4 coordinates. A pseudoenzyme normally shows a reference triad position landing on a non-catalytic residue or in a gap.

• References above the computed 31.3% identity cut - the rows where a global alignment places single residues reliably - whose full annotated triad projects onto AADACL4 residues [193, 347, 377] with identical residue type: 7 of 7.
• The cut is applied to the computed identity, not to a hand-labelled set, and the run aborts if any member lands within 0.75 point of it. Whether the numeric cut reproduces AADAC-family membership exactly: True.
• The remaining rows sit at 25-30% identity, where the alignment rather than the biology decides where an individual position lands; they are shown for completeness and carry no weight in the register claim. One of them is informative anyway: soybean HIDH's annotated nucleophile is a threonine, not a serine, which is the single clearest sign that this node is not a uniform serine-esterase family.

Part 3 - full audit of the hydrolase-activity IBA WITH/FROM column

GO:0016787 hydrolase activity (IBA, GO_REF:0000033) was propagated from PANTHER node PTN009058710. Every token in that GOA row's WITH/FROM column is resolved below and classified by the EC number UniProt assigns to it.

Each non-PANTHER token's resolution is checked back against the resolved entry's own cross-references before anything is reported, and a mismatch aborts the run; 19 token resolutions across both IBA rows passed.

Read-out

• ester hydrolase (EC 3.1.-): 9
• no EC assigned: 4
• amide hydrolase (EC 3.5.-): 2
• ester hydrolase (EC 3.1.-) + lyase (EC 4.-): 1

Part 4 - the N-terminal call, and whether the membrane-IBA donors agree

AADACL4's two GO:0016020 membrane rows both trace to a predicted N-terminal signal anchor. This tabulates what UniProt says about the equivalent segment in the closest paralogs and in the characterised relatives, with each feature's evidence code.

Limitation, stated plainly. Mean hydropathy cannot discriminate a cleaved signal peptide from an uncleaved type-II signal anchor - that is precisely why both calls in this family carry ECO:0000255. No licensed predictor (SignalP, Phobius, TMHMM, DeepTMHMM) was run here, so the hydropathy columns are reported as a like-for-like comparison only and no cleavage-site claim is made from them.

Do the membrane-IBA donors agree on which membrane?

These are the tokens in the WITH/FROM column of the GO:0016020 membrane IBA row, with the subcellular locations UniProt records for each.

• Distinct specific locations across the donors: 4 (Cell membrane, Endoplasmic reticulum membrane, Microsome, Microsome membrane).

Interpretation

Part 1 settles the question the GO record turns on. AADACL4 carries Ser193-Asp347-His377 with the nucleophile sitting in a GESVG elbow and the HGG oxyanion-hole motif upstream. The catalytic machinery of the GDXG lipolytic-enzyme family is complete, so an ester-hydrolase molecular function is a homology inference about an intact active site, not a fold name transcribed into an activity. Equally, the fold-without-function reading is not available: there is no lost triad residue and no displaced elbow to point at.

Part 2 shows the same thing without relying on position numbers: the annotated triads of the AADAC-family relatives project onto exactly the three AADACL4 residues UniProt annotates, at the residue identities expected.

Part 3 explains why the phylogenetic annotation stops at the general hydrolase activity parent instead of naming an ester hydrolase. The node it was propagated from reaches back past the plant/fungal/bacterial split and mixes ester hydrolases with two arylformamidases (EC 3.5.1.9, an amide bond) and a 2-hydroxyisoflavanone dehydratase that is also classified as a lyase. At that depth hydrolase activity is the correct last-common-ancestor call, so the general term is not an over-general slip - it is just less informative than what the protein's own subfamily signature supports.

Part 4 records the soft spot. Within a set of paralogs that share the catalytic register exactly, UniProt's N-terminal calls diverge - signal anchor for AADACL4, a cleaved signal peptide for AADACL2, nothing at all for AADACL3 despite a comparably hydrophobic segment - and every one of those calls is a sequence-analysis prediction. The membrane-IBA donors then disagree about which membrane, so the general membrane term is also the correct call rather than a lazy one: refining it would require picking one donor over the other.

What none of this establishes is a substrate. An intact GDXG active site is compatible with any carboxylic ester, so no substrate-level or compartment-specific term is licensed by homology alone.