ManX (also known as EIIAB-Man or EIII-Man) is the cytoplasmic phosphorylating subunit of the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system (Man-PTS) in E. coli K12. It is a bifunctional protein containing two PTS domains: an N-terminal EIIA domain (phosphorylated by HPr at His-10) and a C-terminal EIIB domain (phosphorylated at His-175), connected by a flexible hinge region. ManX forms homodimers and functions together with the integral membrane subunits ManY (EIIC) and ManZ (EIID) to transport and phosphorylate mannose, glucose, 2-deoxyglucose, fructose, and N-acetylglucosamine via PEP-dependent phosphorylation. ManX exists both free in the cytoplasm and associated with the inner membrane. The ManXYZ complex also serves as a receptor for bacteriophage lambda DNA injection and as a chemoreceptor for sugars in bacterial chemotaxis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0008982 protein-N(PI)-phosphohistidine-sugar phosphotransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for phosphohistidine-sugar phosphotransferase activity. ManX is the EIIAB component of the mannose PTS and is directly phosphorylated at His-10 (by HPr) and His-175 (EIIA to EIIB transfer), then transfers the phosphoryl group to the sugar substrate (PMID:2681202, PMID:8262947). This is a well-supported core molecular function. The IBA term GO:0008982 uses the generic "carbohydrate" parent; the more specific GO:0022870 (mannose-specific) is also annotated. Both are appropriate at their respective levels. Reason: This IBA annotation correctly captures the core phosphotransferase activity of ManX at an appropriate phylogenetic level. ManX catalyzes PEP-dependent sugar phosphorylation as documented by Erni et al. (PMID:2951378, PMID:2999119) and confirmed by mutagenesis studies (PMID:8262947). The IBA is consistent with the experimentally supported annotations. Supporting Evidence: PMID:2951378 IIIMan (35 kDa) is a hydrophilic protein which is transiently phosphorylated and most likely contains the active site for sugar phosphorylation PMID:2999119 IIIMan can be phosphorylated in a phosphoenolpyruvate-dependent reaction |
| GO:0009401 phosphoenolpyruvate-dependent sugar phosphotransferase system | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for involvement in PEP-dependent sugar PTS. ManX is an essential component of the mannose PTS (PMID:2951378, PMID:2999119). This is a core biological process. Reason: Core biological process for ManX. The mannose permease mediates sugar transport via the PEP-dependent PTS as shown by multiple studies (PMID:2951378, PMID:2999119). The IBA is phylogenetically sound and experimentally well supported. Supporting Evidence: PMID:2951378 The mannose permease of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation PMID:2999119 The mannose-permease complex of the phosphoenolpyruvate-dependent phosphotranferase system exhibits two apparently unrelated activities |
| GO:1902495 transmembrane transporter complex | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for localization to a transmembrane transporter complex. ManX associates with the ManY/ManZ integral membrane components to form the ManXYZ transporter complex (PMID:2951378, PMID:2999119). ManX is the peripheral cytoplasmic subunit of this complex. Reason: ManX forms a complex with the transmembrane subunits ManY and ManZ. While ManX itself is soluble/peripheral, it is part of the transmembrane transporter complex. Erni & Zanolari showed IIMan and IIIMan form a complex (PMID:2999119) and the full permease consists of three subunits (PMID:2951378). ComplexPortal CPX-5968 documents the D-mannose-specific enzyme II complex. Supporting Evidence: PMID:2951378 The permease consists of three different subunits, IIIMan, II-PMan, and II-MMan, which are encoded in a single transcriptional unit ptsLPM PMID:2999119 A complex of two proteins, IIMan and IIIMan, was purified to homogeneity from an overproducing strain file:ECOLI/manX/manX-deep-research-falcon.md Multiple sources explicitly place **manX** with **manY** and **manZ** in the **manXYZ** locus, where manY and manZ encode the membrane permease subunits. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation for cytoplasm localization. This is redundant with the IDA annotation for cytoplasm (PMID:2999119) but is not incorrect. ManX (IIIMan) exists both membrane-associated and free in the cytoplasm (PMID:2999119). Reason: Correct IEA annotation, consistent with experimental evidence. ManX is found both in the cytoplasm and membrane-associated (PMID:2999119). The IDA annotation with the same GO ID provides stronger evidence for the same localization. Supporting Evidence: PMID:2999119 IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation for plasma membrane localization based on UniProt subcellular location mapping. Consistent with the IDA annotation for the same term (PMID:2999119). Reason: Correct. ManX is a peripheral membrane protein associated with the cell inner membrane (PMID:2999119). Redundant with the IDA-supported annotation but not incorrect. Supporting Evidence: PMID:2999119 IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm |
| GO:0008643 carbohydrate transport | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation for carbohydrate transport based on InterPro domain mapping. ManX is involved in transport of mannose, glucose, fructose, and N-acetylglucosamine (PMID:2951378, PMID:2999119). This is a broad parent term; more specific sugar transport terms are also annotated. Reason: Correct but general. ManX participates in the transport of multiple sugars including mannose, glucose, fructose, and N-acetylglucosamine. More specific transport annotations exist (GO:0015761, GO:0098708, GO:1990539, GO:0015764) but this broader IEA term is not wrong. Supporting Evidence: PMID:2951378 The mannose permease of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation |
| GO:0008982 protein-N(PI)-phosphohistidine-sugar phosphotransferase activity | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation for phosphohistidine-sugar phosphotransferase activity from InterPro domain mapping. Redundant with the IBA annotation for the same GO term. Correct. Reason: Correct IEA annotation consistent with the IBA and experimentally supported function. ManX contains PTS EIIA and EIIB domains that mediate phosphotransfer (PMID:2681202, PMID:8262947). |
| GO:0009401 phosphoenolpyruvate-dependent sugar phosphotransferase system | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation for PEP-dependent sugar PTS based on InterPro. Redundant with IBA and IDA annotations for the same GO term. Correct. Reason: Correct IEA annotation. ManX is a core component of the PEP-dependent sugar PTS (PMID:2951378, PMID:2999119). Duplicates more authoritative evidence codes for the same term. |
| GO:0016020 membrane | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation for membrane localization from InterPro. This is a very broad CC term. ManX is a peripheral membrane protein (PMID:2999119). The more specific GO:0005886 (plasma membrane) is also annotated. Reason: Not incorrect but very general. ManX associates with the inner membrane as a peripheral membrane protein (PMID:2999119). More specific terms (GO:0005886) are also present. Acceptable as a broad IEA annotation. |
| GO:0016773 phosphotransferase activity, alcohol group as acceptor | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation for phosphotransferase activity (alcohol group acceptor) from InterPro. ManX transfers phosphoryl groups to the C6 hydroxyl of mannose (an alcohol group), so this is technically correct. However, the more specific GO:0008982 (protein-N(PI)-phosphohistidine-carbohydrate phosphotransferase activity) and GO:0022870 (mannose-specific PTS transporter activity) are better descriptors. Reason: Correct but very general IEA annotation. ManX phosphorylates sugar substrates at hydroxyl groups. More specific MF terms are already annotated. Acceptable as a broad IEA mapping. |
| GO:0005515 protein binding | IPI PMID:16606699 Large-scale identification of protein-protein interaction of... | REMOVE | Summary: IPI annotation for protein binding based on large-scale pull-down assay (PMID:16606699). The interacting partner is CytR (P0ACN7) per the GOA WITH column. Per curation guidelines, GO:0005515 (protein binding) is uninformative and should be removed. ManX has well-characterized specific interactions (homodimerization, interaction with HPr) that are captured by more specific terms. Reason: Per curation guidelines, "protein binding" (GO:0005515) is uninformative and does not convey meaningful functional information about ManX. The large-scale pull-down study (PMID:16606699) identifies interactions but the generic "protein binding" term does not add useful annotation. More specific interaction terms like GO:0042803 (protein homodimerization activity) are already annotated. Supporting Evidence: PMID:16606699 A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library |
| GO:0009401 phosphoenolpyruvate-dependent sugar phosphotransferase system | IDA PMID:2951378 The mannose permease of Escherichia coli consists of three d... | ACCEPT | Summary: IDA annotation for PEP-dependent sugar PTS based on the seminal Erni et al. 1987 paper that characterized the mannose permease as consisting of three subunits mediating sugar transport and phosphorylation via the PTS (PMID:2951378). Reason: Core biological process. Erni et al. 1987 demonstrated that the mannose permease mediates PEP-dependent sugar transport and phosphorylation (PMID:2951378). This is the primary function of ManX. Supporting Evidence: PMID:2951378 The mannose permease of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation |
| GO:0015761 mannose transmembrane transport | NAS PMID:2951378 The mannose permease of Escherichia coli consists of three d... | ACCEPT | Summary: NAS annotation for mannose transmembrane transport. Erni et al. 1987 explicitly described the mannose permease as mediating mannose transport (PMID:2951378). ManX is the EIIAB component required for mannose transport and phosphorylation. Reason: Mannose transport is the primary named function of the ManXYZ complex. Although ManX alone is not the transmembrane component (ManY/ManZ form the channel), ManX is essential for the transport-coupled phosphorylation. PMID:2951378 states all three subunits are required for sugar transport. Supporting Evidence: PMID:2951378 All three subunits are required for sugar transport and phosphorylation |
| GO:0015761 mannose transmembrane transport | IDA PMID:5545083 Sugar transport. II. Characterization of constitutive membra... | ACCEPT | Summary: IDA annotation for mannose transmembrane transport based on Kundig & Roseman 1971 (PMID:5545083), which characterized constitutive membrane-bound enzymes II of the E. coli PTS including the mannose-specific system. Reason: This early characterization paper established the mannose-specific PTS enzyme II activity in E. coli membranes. ManX is essential for mannose transport by the ManXYZ complex. Supporting Evidence: PMID:5545083 Sugar transport. II. Characterization of constitutive membrane-bound enzymes II of the Escherichia coli phosphotransferase system |
| GO:0015764 N-acetylglucosamine transport | EXP PMID:6252281 Amino-sugar transport systems of Escherichia coli K12. | KEEP AS NON CORE | Summary: EXP annotation for N-acetylglucosamine transport. Jones-Mortimer & Kornberg 1980 (PMID:6252281) showed that N-acetylglucosamine enters E. coli by two PTS systems, one of which is the PtsM (ManXYZ) system. This is a secondary transport substrate of the Man-PTS. Reason: N-acetylglucosamine transport via the Man-PTS is experimentally documented (PMID:6252281) but represents a secondary substrate rather than the core mannose/glucose transport function. The Man-PTS has broad substrate specificity. Supporting Evidence: PMID:6252281 N-Acetylglucosamine enters E. coli by two distinct phosphotransferase systems file:ECOLI/manX/manX-deep-research-falcon.md the mannose-family PTS can be **promiscuous** in substrate handling beyond mannose |
| GO:0098708 D-glucose import across plasma membrane | IDA PMID:5545083 Sugar transport. II. Characterization of constitutive membra... | KEEP AS NON CORE | Summary: IDA annotation for D-glucose import across plasma membrane. The Man-PTS is known to transport glucose in addition to mannose (PMID:2999119 mentions 2-deoxyglucose transport). Glucose is an important substrate of the Man-PTS. Reason: Glucose transport by the Man-PTS is well established. Erni & Zanolari 1985 showed the mannose permease mediates transport of mannose, 2-deoxyglucose, and other hexoses (PMID:2999119). However, glucose transport is a secondary function; the primary named substrate is mannose. Supporting Evidence: PMID:2999119 It mediates active transport concomitant with phosphorylation of mannose, 2-deoxyglucose, and a number of other hexoses file:ECOLI/manX/manX-deep-research-falcon.md lists substrates associated with the ManXYZ PTS system including **mannose, glucose, 2-deoxyglucose, fructose**, and amino sugars (including **GlcNAc/GlcN**) |
| GO:1990539 fructose import across plasma membrane | EXP PMID:4153999 The role of phosphotransferase-mediated syntheses of fructos... | KEEP AS NON CORE | Summary: EXP annotation for fructose import across plasma membrane. Ferenci & Kornberg 1974 (PMID:4153999) studied PTS-mediated fructose phosphorylation in E. coli. The Man-PTS can transport fructose as a secondary substrate. Reason: Fructose import via the Man-PTS is a secondary transport function. E. coli has a dedicated fructose PTS (FruAB), and fructose transport via the Man-PTS is a minor pathway. This is a legitimate but non-core annotation. Supporting Evidence: PMID:4153999 The role of phosphotransferase-mediated syntheses of fructose 1-phosphate and fructose 6-phosphate in the growth of Escherichia coli on fructose |
| GO:1990539 fructose import across plasma membrane | EXP PMID:4154035 Genetical analysis of fructose utilization by Escherichia co... | KEEP AS NON CORE | Summary: EXP annotation for fructose import based on Jones-Mortimer & Kornberg 1974 (PMID:4154035), a genetic analysis of fructose utilization showing the Man-PTS can participate in fructose uptake. Reason: Duplicate of the fructose import annotation with a different reference. Fructose transport is a secondary substrate for the Man-PTS. Keeping as non-core. Supporting Evidence: PMID:4154035 Genetical analysis of fructose utilization by Escherichia coli |
| GO:0005829 cytosol | HDA PMID:16858726 A complexomic study of Escherichia coli using two-dimensiona... | ACCEPT | Summary: HDA annotation for cytosol localization based on complexomic study using 2D BN/SDS-PAGE (PMID:16858726). ManX was identified in the cytosolic fraction. Consistent with its known dual localization (cytoplasm and membrane-associated). Reason: ManX is found in the cytosol as established by multiple approaches. The complexomic study (PMID:16858726) identified ManX in both cytosolic and membrane fractions, consistent with the biochemical evidence (PMID:2999119). Supporting Evidence: PMID:16858726 the cytosolic and membrane protein complexes of Escherichia coli were separated PMID:2999119 IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm |
| GO:0005737 cytoplasm | IDA PMID:2999119 The mannose-permease of the bacterial phosphotransferase sys... | ACCEPT | Summary: IDA annotation for cytoplasm localization. Erni & Zanolari 1985 (PMID:2999119) showed that IIIMan (ManX) exists both membrane-associated and free in the cytoplasm. Reason: Well-supported localization. ManX is a soluble/peripheral protein found in the cytoplasm (PMID:2999119). Supporting Evidence: PMID:2999119 IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm |
| GO:0005829 cytosol | IDA PMID:2999119 The mannose-permease of the bacterial phosphotransferase sys... | ACCEPT | Summary: IDA annotation for cytosol localization based on Erni & Zanolari 1985 (PMID:2999119). ManX is found free in the cytoplasm/cytosol when not membrane-associated. Reason: Consistent with the biochemical purification showing ManX exists as a soluble cytoplasmic protein (PMID:2999119). Cytosol is a reasonable specific localization for the free form. Supporting Evidence: PMID:2999119 IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm file:ECOLI/manX/manX-deep-research-falcon.md ManX is annotated as **IM, C**, consistent with a **cytosolic/peripheral phosphotransfer component associated with the inner membrane complex**. |
| GO:0005886 plasma membrane | IDA PMID:2999119 The mannose-permease of the bacterial phosphotransferase sys... | ACCEPT | Summary: IDA annotation for plasma membrane localization. Erni & Zanolari 1985 (PMID:2999119) showed ManX is found membrane-associated. UniProt confirms it is a peripheral membrane protein of the cell inner membrane. Reason: Well-supported localization. ManX associates with the inner (plasma) membrane as a peripheral membrane protein (PMID:2999119). This represents one of its two localizations (cytoplasmic and membrane-associated). Supporting Evidence: PMID:2999119 IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm |
| GO:0009401 phosphoenolpyruvate-dependent sugar phosphotransferase system | IDA PMID:2999119 The mannose-permease of the bacterial phosphotransferase sys... | ACCEPT | Summary: IDA annotation for PEP-dependent sugar PTS based on Erni & Zanolari 1985 (PMID:2999119). This paper demonstrated PEP-dependent phosphorylation of IIIMan and reconstituted phosphotransferase activity in vitro. Reason: Core biological process. The paper directly demonstrated PEP-dependent phosphorylation of ManX and reconstituted sugar phosphorylation activity (PMID:2999119). Supporting Evidence: PMID:2999119 IIIMan can be phosphorylated in a phosphoenolpyruvate-dependent reaction |
| GO:0016301 kinase activity | IDA PMID:2999119 The mannose-permease of the bacterial phosphotransferase sys... | MARK AS OVER ANNOTATED | Summary: IDA annotation for kinase activity. ManX phosphorylates sugar substrates, which is a kinase activity. However, this is a very broad term. The more specific GO:0008982 (protein-N(PI)-phosphohistidine-carbohydrate phosphotransferase activity) and GO:0022870 (mannose-specific PTS transporter activity) better capture this function. Reason: While ManX does have phosphotransferase (kinase-like) activity, GO:0016301 is too broad and does not capture the PTS-specific mechanism. ManX is not a conventional kinase; it transfers phosphoryl groups from phosphohistidine intermediates to sugars as part of the PTS system. The more specific terms GO:0008982 and GO:0022870 are already annotated and better represent this function. GO:0016301 could be misleading. Supporting Evidence: PMID:2999119 IIMan and IIIMan are both required for phosphorylation of 2-deoxyglucose in vitro |
| GO:0022870 protein-N(PI)-phosphohistidine-mannose phosphotransferase system transporter activity | IDA PMID:5545083 Sugar transport. II. Characterization of constitutive membra... | ACCEPT | Summary: IDA annotation for mannose-specific PTS transporter activity. This is the most specific molecular function term for ManX, capturing both the mannose substrate specificity and the PTS phosphotransferase mechanism. ManX is the EIIAB phosphorylating subunit of the mannose-specific PTS (PMID:2951378, PMID:2999119). Reason: This is the most informative and specific MF term for ManX. It captures the mannose-specific PTS transporter activity that is the core molecular function of the ManXYZ complex. Well supported by multiple studies (PMID:2951378, PMID:2999119, PMID:2681202, PMID:8262947). Supporting Evidence: PMID:2951378 The mannose permease of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation PMID:2999119 It mediates active transport concomitant with phosphorylation of mannose, 2-deoxyglucose, and a number of other hexoses file:ECOLI/manX/manX-deep-research-falcon.md ManX** is the **mannose-family PTS EIIAB subunit**. In curated annotations, it is associated with the inner-membrane system but is itself annotated as a **cytosolic component (IM, C)**, consistent with a cytosolic phosphotransfer function that operates in conjunction with inner-membrane permease components. |
| GO:0042803 protein homodimerization activity | IDA PMID:2951378 The mannose permease of Escherichia coli consists of three d... | ACCEPT | Summary: IDA annotation for protein homodimerization activity. ManX (IIIMan) forms homodimers as demonstrated by Erni et al. 1987 (PMID:2951378) and confirmed by Erni & Zanolari 1985 (PMID:2999119) and structural studies. UniProt states "Homodimer" with multiple supporting references. Reason: Well-supported molecular function. ManX forms stable homodimers, confirmed by biochemical purification (PMID:2999119), characterization studies (PMID:2951378), and NMR structural studies. Dimerization is functionally relevant as shown by mutagenesis (His-10 and other mutations abolish dimerization; PMID:8262947). Supporting Evidence: PMID:2999119 IIIMan, a 35-kDa protein, exists as a dimer PMID:2951378 IIIMan (35 kDa) is a hydrophilic protein which is transiently phosphorylated |
| GO:0016020 membrane | HDA PMID:16858726 A complexomic study of Escherichia coli using two-dimensiona... | ACCEPT | Summary: HDA annotation for membrane localization based on the complexomic study (PMID:16858726). ManX was found in both cytosolic and membrane fractions. This is a broad CC term; the more specific GO:0005886 (plasma membrane) is also annotated. Reason: Correct but broad. The complexomic study identified ManX in membrane fractions (PMID:16858726), consistent with its known peripheral membrane association (PMID:2999119). The more specific plasma membrane term is also annotated. Supporting Evidence: PMID:16858726 the cytosolic and membrane protein complexes of Escherichia coli were separated |
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Download this section (compressed HTML)Q: Should the chemoreceptor function of the Man-PTS (sugar chemotaxis) be annotated for ManX specifically, or is this a property of the complex? Adler & Epstein 1974 (PMID:4604906) showed PTS enzymes serve as chemoreceptors.
Q: Should the role in bacteriophage lambda DNA injection be annotated? The pel mutant phenotype maps to the Man-PTS (PMID:353494), but ManY/ManZ (not ManX) are sufficient for lambda DNA penetration (PMID:2951378).
Q: Is the GO:0016301 (kinase activity) annotation appropriate for a PTS phosphotransferase, or is this misleading given that the mechanism involves phosphohistidine intermediates rather than conventional kinase activity?
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