PedH is a lanthanide-dependent, pyrroloquinoline quinone (PQQ)-using alcohol dehydrogenase of Pseudomonas putida KT2440. It acts in the periplasm on diverse primary and secondary alcohols, including aromatic substrates, and also oxidizes some aldehydes. Together with the alternative metal-dependent paralog it provides condition-dependent entry into (2S,3S)-2,3-butanediol utilization. The PedS2/PedR2 regulatory system adjusts expression to lanthanide availability. A cytochrome c acceptor is assigned by enzyme-family homology; the specific physiological KT2440 electron-transfer partner remains unresolved. PedH also contributes a positive expression-feedback phenotype whose signaling mechanism is unresolved.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005509 calcium ion binding | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: Calcium supports PedE catalysis, whereas PedH uses lanthanides under the tested conditions. Reason: This electronic calcium assignment overextends the calcium-dependent quinoprotein model to the lanthanide-dependent PedH active site. Purified PedH is inactive under the calcium conditions that activate PedE, but active with tested early lanthanides; the PedH 6ZCW structure contains praseodymium and PQQ. These observations establish the functional metal distinction, not an absolute inability to bind calcium nonspecifically. Supporting Evidence: PMID:28655819 When we assayed purified PedH under the optimized reaction conditions used for PedE, no activity was observed with any of the substrates tested (data not shown). PMID:28655819 However, in the presence of the lanthanide La3+, Ce3+, Pr3+, Nd3+, Sm3+, Gd3+, or Tb3+ at 1 µM, enzymatic activity was detected, with maximal specific activity observed with Pr3+ and Nd3+ and only very low activity observed with Gd3+ and Tb3+. |
| GO:0016020 membrane | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: These soluble type I quinoproteins act in the periplasm; a membrane-bound role is not established. Reason: The mapping to membrane overextends the broader PQQ-dehydrogenase group, which includes membrane-bound and soluble classes. These target proteins are classified as type I quinoproteins and perform periplasmic oxidation; soluble recombinant purification agrees with that assignment. The membrane claim is not established by these observations, but neither absence of a transmembrane helix nor purification alone excludes transient peripheral membrane contact. Supporting Evidence: PMID:30158283 In a recent study, we found that these two type I quinoproteins (9, 10) exhibit a similar substrate scope but require different metal cofactors (8). PMID:30158283 For efficient capture and metabolism of such VOCs, P. putida makes use of two pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenases (PQQ-EDHs)—namely, PedE and PedH—to carry out the initial oxidation steps in the periplasm of the cell (7, 8). PMID:28655819 A one-step affinity chromatography method produced soluble C-terminally His-tagged PedE and PedH to visible purity (see Fig. S1 in the supplemental material) from cell lysates of Escherichia coli BL21(DE3). |
| GO:0016614 oxidoreductase activity, acting on CH-OH group of donors | IEA GO_REF:0000002 | ACCEPT | Summary: Primary and secondary alcohol oxidation are both supported catalytic activities. Reason: Retain this enzyme-class activity as core because it covers the demonstrated primary and secondary alcohol substrates. GO:0052934 additionally represents the inferred cytochrome-c-dependent primary-alcohol branch, but does not cover secondary-alcohol-to-ketone chemistry such as (2S,3S)-butanediol entry. The broad term is therefore needed to preserve the supported substrate scope. Supporting Evidence: PMID:28655819 Similarly, 2-phenylethanol, the secondary alcohol 2-butanol, cinnamyl alcohol, and the acyclic sesquiterpene farnesol were efficiently converted, with specific activities ranging from 6.7 ± 1.1 to 2.0 ± 0.3 U mg−1. PMID:34513815 Double-knockout strain P. putida KT2440 (ΔpedEΔpedH) was completely unable to utilize (2S,3S)-2,3-BDO both in the presence and absence of La3+ (Figures 5C,D) |
| GO:0030288 outer membrane-bounded periplasmic space | IEA GO_REF:0000002 | ACCEPT | Summary: The native oxidation system acts in the outer membrane-bounded periplasm. Reason: The native KT2440 oxidation system acts in the periplasm of a bacterium with an outer membrane. This term denotes the compartment bounded by that membrane; it does not require enzyme attachment to the outer membrane. The earlier contrary interpretation is superseded. Supporting Evidence: PMID:30158283 For efficient capture and metabolism of such VOCs, P. putida makes use of two pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenases (PQQ-EDHs)—namely, PedE and PedH—to carry out the initial oxidation steps in the periplasm of the cell (7, 8). |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000120 | MODIFY | Summary: Alcohol and aldehyde oxidation define the supported oxidoreductase activities. Reason: Refine the correct general oxidoreductase activity to the already represented CH-OH donor activity GO:0016614. Retaining that breadth matters: experimentally supported secondary-alcohol oxidation to ketones is not entailed by the primary-alcohol-to-aldehyde definition of GO:0052934. Also retain the independently measured aldehyde-donor branch through GO:0016903; CH-OH activity alone would lose the acetaldehyde and other aldehyde oxidation demonstrated in Table 1. Proposed replacements: oxidoreductase activity, acting on CH-OH group of donors oxidoreductase activity, acting on the aldehyde or oxo group of donors Supporting Evidence: PMID:28655819 Similarly, 2-phenylethanol, the secondary alcohol 2-butanol, cinnamyl alcohol, and the acyclic sesquiterpene farnesol were efficiently converted, with specific activities ranging from 6.7 ± 1.1 to 2.0 ± 0.3 U mg−1. PMID:34513815 Double-knockout strain P. putida KT2440 (ΔpedEΔpedH) was completely unable to utilize (2S,3S)-2,3-BDO both in the presence and absence of La3+ (Figures 5C,D) PMID:28655819 Under optimized conditions, which included supplementation with 1 µM Pr3+ instead of Ca2+, PedH showed an activity pattern similar to that of PedE (Table 1) but exhibited about 2-fold higher specific activity. |
| GO:0020037 heme binding | IEA GO_REF:0000117 | REMOVE | Summary: PQQ, rather than a heme prosthetic group, is the bound redox cofactor. Reason: The target is a type I quinoprotein, not a heme-containing quinohemoprotein. Its bound redox cofactor is PQQ; a separate cytochrome electron acceptor does not imply a heme prosthetic group within this enzyme. The ARBA-derived heme assignment conflates those enzyme classes. Supporting Evidence: PMID:30158283 In a recent study, we found that these two type I quinoproteins (9, 10) exhibit a similar substrate scope but require different metal cofactors (8). PMID:28655819 The reaction mechanism of PQQ-ADHs is still not completely resolved but most likely proceeds via an ion-assisted direct hydride transfer from the substrate to C-5 of the noncovalently bound cofactor PQQ (10–12). |
| GO:0042597 periplasmic space | IEA GO_REF:0000120 | MODIFY | Summary: The native periplasm is specifically the outer membrane-bounded compartment. Reason: Refine the general periplasmic-space term to the already represented outer membrane-bounded periplasmic space GO:0030288, appropriate for the native Gram-negative organism. This is a compartment specification, not a claim of membrane binding or a whole-organelle replacement. Proposed replacements: outer membrane-bounded periplasmic space Supporting Evidence: PMID:30158283 For efficient capture and metabolism of such VOCs, P. putida makes use of two pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenases (PQQ-EDHs)—namely, PedE and PedH—to carry out the initial oxidation steps in the periplasm of the cell (7, 8). |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | ACCEPT | Summary: PedH binds catalytic lanthanides, including structurally observed praseodymium. Reason: The experimentally supported bound catalytic metals are lanthanides. Metal ion binding is retained as the available GO class without implying that all metals activate the enzyme. Activity is strongest with Pr and Nd in the tested panel; Gd and Tb have only low activity. The 6ZCW deposition independently identifies bound praseodymium. Supporting Evidence: PMID:28655819 However, in the presence of the lanthanide La3+, Ce3+, Pr3+, Nd3+, Sm3+, Gd3+, or Tb3+ at 1 µM, enzymatic activity was detected, with maximal specific activity observed with Pr3+ and Nd3+ and only very low activity observed with Gd3+ and Tb3+. |
| GO:0052934 alcohol dehydrogenase (cytochrome c) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Primary alcohol oxidation is measured, with the cytochrome c acceptor assigned by enzyme-family inference. Reason: Accept the primary-alcohol enzyme-class assignment by measured alcohol oxidation plus curated ExaA-family electron-acceptor inference. The 2017 assays use artificial PMS/DCPIP and do not identify the native KT2440 cytochrome c partner. The GO definition is primary alcohol to aldehyde: this term is not used to stand for secondary butanediol oxidation to acetoin. Supporting Evidence: PMID:28655819 Similarly, 2-phenylethanol, the secondary alcohol 2-butanol, cinnamyl alcohol, and the acyclic sesquiterpene farnesol were efficiently converted, with specific activities ranging from 6.7 ± 1.1 to 2.0 ± 0.3 U mg−1. file:PSEPK/pedH/pedH-uniprot.txt Uses a specific CC inducible cytochrome c550, encoded by the adjacent gene in the locus, CC as electron acceptor (By similarity). |
| GO:0070968 pyrroloquinoline quinone binding | IEA GO_REF:0000117 | ACCEPT | Summary: PQQ binding supplies the redox cofactor for catalysis. Reason: PQQ is the enzyme redox cofactor. Target-protein activity measurements require added PQQ; curated cofactor and structural evidence support binding independently of the electronic annotation. PedH also has directly deposited PQQ in 6ZCW. No inference of heme binding follows. Supporting Evidence: PMID:28655819 The reaction mechanism of PQQ-ADHs is still not completely resolved but most likely proceeds via an ion-assisted direct hydride transfer from the substrate to C-5 of the noncovalently bound cofactor PQQ (10–12). file:PSEPK/pedH/pedH-uniprot.txt Binds 1 PQQ group per subunit. |
| GO:0006066 alcohol metabolic process | IC PMID:28655819 Functional Role of Lanthanides in Enzymatic Activity and Tra... | NEW | Summary: Direct alcohol oxidation supports native alcohol catabolism. Reason: This is an inherited proposal, not a newly added annotation. Retain because the enzyme itself catalyzes alcohol oxidation, supported by purified-protein substrate measurements and native catabolic phenotypes. It is not inferred solely from knockout necessity. The broader process includes the condition-dependent secondary-butanediol entry, so no redundant new butanediol process is proposed. The author-controlled proposal uses IC from measured target-enzyme turnover, replacing its inherited IEA placeholder; this does not edit a GOA assertion. Supporting Evidence: PMID:28655819 Similarly, 2-phenylethanol, the secondary alcohol 2-butanol, cinnamyl alcohol, and the acyclic sesquiterpene farnesol were efficiently converted, with specific activities ranging from 6.7 ± 1.1 to 2.0 ± 0.3 U mg−1. PMID:34513815 Double-knockout strain P. putida KT2440 (ΔpedEΔpedH) was completely unable to utilize (2S,3S)-2,3-BDO both in the presence and absence of La3+ (Figures 5C,D) |
| GO:0042537 benzene-containing compound metabolic process | IC PMID:28655819 Functional Role of Lanthanides in Enzymatic Activity and Tra... | NEW | Summary: Direct aromatic-alcohol oxidation participates in benzene-containing compound metabolism. Reason: Retain this inherited proposal for direct oxidation of benzene-containing alcohols, including 2-phenylethanol and cinnamyl alcohol. Purified KT2440 protein chemistry establishes participation, rather than assigning a process solely from a growth defect or from the name of the Ped pathway in strain U. The author-controlled proposal uses IC from measured target-enzyme turnover, replacing its inherited IEA placeholder; this does not edit a GOA assertion. Supporting Evidence: PMID:28655819 Similarly, 2-phenylethanol, the secondary alcohol 2-butanol, cinnamyl alcohol, and the acyclic sesquiterpene farnesol were efficiently converted, with specific activities ranging from 6.7 ± 1.1 to 2.0 ± 0.3 U mg−1. |
| GO:0010468 regulation of gene expression | IMP PMID:28655819 Functional Role of Lanthanides in Enzymatic Activity and Tra... | MARK AS OVER ANNOTATED | Summary: PedH-dependent expression feedback is measured, but PedH's own regulatory participation is not shown; over-annotation. Reason: Withdrawn proposal, kept as a retired row and marked over-annotated. Native promoter reporters show PedH-dependent positive feedback on pedH expression, and a later study proposes a lanthanide-sensor role. That is necessity evidence, though, and an essential enzyme can act indirectly through a metabolite it produces. No experiment shows PedH itself doing regulatory work, such as contacting the regulatory machinery, so asserting a regulation-of-gene-expression process overstates the evidence. The feedback phenotype itself is not disputed. The retired flag records that this was an earlier proposal absent from GOA, not a historical GOA annotation. Supporting Evidence: PMID:28655819 Notably, the expression from the pedH promoter was dramatically reduced (2-fold versus 37-fold) in the ΔpedH strain in comparison with that in the strains capable of producing a functional PedH protein. PMID:30158283 In addition, the transcription of pedH is activated via a yet unknown pathway, in which a functional PedH protein is an essential component, most likely by acting as a lanthanide sensor (8). |
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Download this section (compressed HTML)Q: Which native KT2440 electron-transfer partner reoxidizes the enzyme-bound PQQ, and can that transfer be reconstituted with purified components?
Q: What is the quantitative stereospecificity for (2S,3S)-butanediol versus the other isomers under native metal conditions?
Q: Does PedH provide a direct sensory input to transcription, or does its catalytic chemistry produce the positive-feedback signal?
Q: Would a lanthanide-ion-binding GO term represent the directly observed praseodymium cofactor site more precisely than metal ion binding?
Experiment: Reconstitute electron transfer using candidate native cytochromes and quantify alcohol oxidation, independently of PMS/DCPIP.
Experiment: Compare purified-enzyme kinetics for all three 2,3-butanediol stereoisomers with matched PQQ and metal conditions.
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