CrtI is the phytoene desaturase of the carotenoid-producing enterobacterium Pantoea ananatis (historically Erwinia uredovora), a 492-residue FAD-dependent flavoprotein oxidoreductase of the amine-oxidase (carotenoid/retinoid oxidoreductase) fold. As a single enzyme it carries out the entire conversion of the colourless C40 hydrocarbon 15-cis-phytoene into the pink, fully conjugated all-trans-lycopene, introducing four double bonds through the successive intermediates phytofluene, zeta-carotene and neurosporene and isomerizing the central 15-cis bond to trans in the course of the reaction. Each desaturation abstracts a pair of hydrogens that are passed via the tightly bound FAD to a terminal electron acceptor, which is molecular oxygen under aerobic conditions and can be replaced by quinones anaerobically. FAD is both the redox cofactor and a structural element: the holoenzyme forms cooperatively as the protein associates with the membrane, and apoprotein that binds the membrane without FAD cannot be reactivated afterwards. Rather than being integral, the enzyme sits peripherally, probably monotopically, on the cytoplasmic membrane where its lipophilic substrate resides. CrtI accepts 15-cis-phytoene from the phytoene synthase CrtB and delivers all-trans-lycopene to the lycopene cyclase CrtY, and because its bacterial single-enzyme route substitutes functionally for the four-enzyme poly-cis desaturation pathway of plants and cyanobacteria (PDS, Z-ISO, ZDS, CRTISO), it has become a standard tool for reconstructing carotenoid pathways in heterologous hosts.
Definition: Catalysis of the reaction 15-cis-phytoene + 4 A = all-trans-lycopene + 4 AH2, in which a single flavoprotein introduces four double bonds into phytoene (via phytofluene, zeta-carotene and neurosporene) and isomerizes the central 15-cis bond to trans, producing all-trans-lycopene using a diffusible electron acceptor (EC 1.3.99.31, RHEA:15585). This is the bacterial and fungal CrtI-type route, mechanistically and cofactor-wise distinct from the plant/cyanobacterial plastoquinone-dependent 15-cis-phytoene desaturase (EC 1.3.5.5) that performs only part of the desaturation sequence.
Justification: GO:0016166 phytoene dehydrogenase activity has no children and is therefore used both for the CrtI four-step lycopene-forming reaction (EC 1.3.99.31, RHEA:15585) and for the plant/cyanobacterial single-substrate PDS reaction (EC 1.3.5.5), which are mutually exclusive routes distinguished only by Rhea and taxon. A dedicated child term for the lycopene-forming activity would let GO discriminate the two desaturation systems by function term. The carotene backbone biosynthesis module records the same absence of a CrtI-specific child of GO:0016166.
Parent term: phytoene dehydrogenase activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt-SubCell mapping of the cell-membrane location that UniProt records for this entry with experimental evidence. CrtI is a membrane-peripheral (probably monotopic) protein that carries out its chemistry on the lipophilic, bilayer-associated phytoene substrate, so it is correctly located at the plasma (cytoplasmic) membrane. Reason: UniProt assigns SUBCELLULAR LOCATION Cell membrane with ECO:0000269 experimental evidence from PMID:1400305, the study in which the enzyme was first shown to be a membrane-integrated carotenogenic protein. The 2012 structural study revised the topology from integral to membrane-peripheral and possibly monotopic, but this is a change in how the protein sits on the bilayer, not in which compartment it acts: it still functions at the cytoplasmic membrane. A more precise leaflet-level term such as cytoplasmic side of plasma membrane was considered and rejected because no retrieved source establishes the sidedness in the native bacterium. The GOA-supplied Swiss-Prot subcellular-location term is therefore retained as the best supported localization. Supporting Evidence: file:PANAN/crtI/crtI-uniprot.txt SUBCELLULAR LOCATION: Cell membrane {ECO:0000269|PubMed:1400305}. PMID:1400305 This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state. PMID:22745782 CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. |
| GO:0016117 carotenoid biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO assignment of the module-level carotenoid biosynthetic process from IPR014105, the carotenoid/retinoid oxidoreductase family signature. The enzyme desaturates phytoene to lycopene, a carotenoid, so the process term is correct. Reason: IPR014105 is the carotenoid/retinoid oxidoreductase family entry, and this protein also matches the NCBIfam model TIGR02734 crtI_fam specific to CrtI-type phytoene desaturases, so the family assignment is sound. Lycopene is a carotenoid and this desaturation step is part of carotenoid biosynthesis, making GO:0016117 the correct module-level process for a CrtI-type desaturase. A domain- or family-level rule appropriately outputs a process term at this level of generality, so the electronic call is accepted as-is. Supporting Evidence: file:PANAN/crtI/crtI-uniprot.txt DR NCBIfam; TIGR02734; crtI_fam; 1. file:PANAN/crtI/crtI-uniprot.txt Converts 15-cis-phytoene into all-trans-lycopene via the |
| GO:0016120 carotene biosynthetic process | IDA PMID:1400305 Expression in Escherichia coli, purification, and reactivati... | MODIFY | Summary: Direct assay of the purified recombinant enzyme converting 15-cis-phytoene to trans-lycopene. The demonstrated product is specifically lycopene, so the process is better described by the verified child term GO:1901177 lycopene biosynthetic process than by the parent carotene biosynthetic process. Reason: Fraser et al. purified the desaturase to homogeneity, restored activity after urea solubilization, and showed directly that the isolated enzyme converts 15-cis-phytoene to trans-lycopene. Because the experiment pins the exact product, the process term can be as specific as the evidence: GO:1901177 lycopene biosynthetic process is a QuickGO-verified, non-obsolete descendant of GO:0016120 carotene biosynthetic process and names precisely the carotene made in this reaction. This is a specificity refinement, not a correction; the essence of the annotation is sound. I deliberately did not touch the ontology relationship between GO:0016120 and GO:0016117 (in the current ontology GO:0016120 is not a descendant of GO:0016117), which is not this review's business to change. Proposed replacements: lycopene biosynthetic process Supporting Evidence: PMID:1400305 The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. file:PANAN/crtI/crtI-uniprot.txt Carotenoid biosynthesis; lycopene biosynthesis. |
| GO:0016120 carotene biosynthetic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning assignment of the carotene biosynthetic process term. The enzyme makes the carotene lycopene, so the process is correct at this level of generality. Reason: This electronic row reaches the correct process for a phytoene desaturase. An ARBA family-level rule that stops at carotene biosynthetic process rather than descending to lycopene biosynthetic process is a broader but not wrong call, and the reviewer guidance permits accepting IEAs that are more general than the specific literature-supported term. The specific child term is proposed on the parallel IDA row rather than being forced onto this electronic one. Duplicate GO identifiers under different evidence codes carry independent provenance and are retained. Supporting Evidence: file:PANAN/crtI/crtI-uniprot.txt Carotenoid biosynthesis; lycopene biosynthesis. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO assignment of the root oxidoreductase activity from IPR002937, the generic amine-oxidase (Pfam PF01593) domain shared with monoamine oxidase and protoporphyrinogen IX oxidoreductase. CrtI is indeed a flavoprotein oxidoreductase, so the term is correct though very general. Reason: Correct but uninformatively broad. CrtI has a precise, experimentally supported molecular function, phytoene dehydrogenase activity (GO:0016166), which this review already proposes as the replacement for the GO:0016627 IDA row (PMID:1400305) and which is the appropriate specific replacement for this generic parent too. Applied consistently with the sibling desaturase reviews PDS and ZDS, which MODIFY the identical IPR002937 row. Proposed replacements: phytoene dehydrogenase activity Supporting Evidence: PMID:22745782 apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising file:PANAN/crtI/crtI-uniprot.txt DR InterPro; IPR002937; Amino_oxidase. |
| GO:0016627 oxidoreductase activity, acting on the CH-CH group of donors | IDA PMID:1400305 Expression in Escherichia coli, purification, and reactivati... | MODIFY | Summary: Direct assay of the purified enzyme desaturating 15-cis-phytoene to lycopene, an activity that acts on carbon-carbon bonds. The demonstrated chemistry is more precisely captured by the verified child term GO:0016166 phytoene dehydrogenase activity. Reason: The purified desaturase was shown to dehydrogenate 15-cis-phytoene through to lycopene, which is oxidoreductase chemistry on the CH-CH group of donors, so GO:0016627 is true but one level above the demonstrated activity. GO:0016166 phytoene dehydrogenase activity is a QuickGO-verified, non-obsolete descendant of GO:0016627 (ancestor path GO:0016166 -> GO:0016627 -> GO:0016491) and matches the assayed reaction exactly. Because the IDA pins the substrate and the overall transformation, the term should be as specific as the experiment; MODIFY to the phytoene dehydrogenase activity child. (Note that GO:0016166 has no children specific to the bacterial lycopene-forming EC 1.3.99.31 reaction versus the plant plastoquinone-dependent EC 1.3.5.5 reaction; that granularity gap is recorded under proposed_new_terms.) Proposed replacements: phytoene dehydrogenase activity Supporting Evidence: PMID:1400305 The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. file:PANAN/crtI/crtI-uniprot.txt Converts 15-cis-phytoene into all-trans-lycopene via the PMID:22745782 Right, CRTI-mediated phytoene desaturation encompassing all four desaturation steps and one cis-trans isomerization step to form all-trans-lycopene. |
| GO:0016627 oxidoreductase activity, acting on the CH-CH group of donors | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning assignment of the same CH-CH-donor oxidoreductase class that the enzyme holds by IDA. The electronic call agrees with the direct experimental evidence at a slightly generic level. Reason: This electronic row reaches the correct oxidoreductase class for a CrtI-type desaturase and is concordant with the IDA row for the same term. A family-level rule that stops at the CH-CH-donor level rather than descending to phytoene dehydrogenase activity is broader but not wrong, and broader IEA terms may be accepted. This ACCEPT differs from the MODIFY on the broader GO:0016491/IPR002937 row because that row is the generic root that the specific activity cleanly supersedes, whereas this CH-CH-donor class is only one step above the specific term; the specific child GO:0016166 is proposed on the parallel IDA row rather than duplicated here. Duplicate identifiers under different evidence codes are retained as independent provenance. Supporting Evidence: PMID:1400305 The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. |
| GO:0071949 FAD binding | IDA PMID:1400305 Expression in Escherichia coli, purification, and reactivati... | ACCEPT | Summary: Direct experimental evidence that FAD is the cofactor of the desaturase. The 2012 structural and reconstitution work independently confirms FAD as the sole redox-active cofactor and shows it to be structurally required for the active holoenzyme, so the FAD-binding activity is firmly grounded. Reason: Fraser et al. established the cofactor role of FAD directly, reporting that FAD was involved in desaturation while NAD and NADP were inhibitory. Schaub et al. reconstituted the enzyme and concluded that FAD is the sole cofactor effective in CRTI-mediated desaturation, and further showed that FAD is required at the moment of membrane association and cannot be added afterwards to rescue apoprotein, so the flavin is both catalytic and structural. UniProt records FAD as the cofactor with experimental evidence. The molecular function is correct and specific and represents a genuine activity of the protein. Supporting Evidence: PMID:22745782 we conclude that FAD is the sole cofactor effective in CRTI-mediated phytoene desaturation PMID:22745782 In contrast, CRTI obtained by membrane association in the absence of FAD was inactive and could not be reactivated by subsequent addition of FAD file:PANAN/crtI/crtI-uniprot.txt Name=FAD; Xref=ChEBI:CHEBI:57692; |
| GO:0071949 FAD binding | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning assignment of the FAD-binding function that the gene already holds by direct experiment. The electronic call agrees with the experimental evidence. Reason: CrtI is an established FAD-dependent flavoprotein, so the ARBA FAD-binding assignment is correct and concordant with the IDA row for the same term. Duplicate GO identifiers under different evidence codes carry independent provenance and are both retained. Supporting Evidence: PMID:22745782 we conclude that FAD is the sole cofactor effective in CRTI-mediated phytoene desaturation file:PANAN/crtI/crtI-uniprot.txt Name=FAD; Xref=ChEBI:CHEBI:57692; |
Loading supporting content…
Download this section (compressed HTML)Q: Which terminal electron acceptor does CrtI use in native Pantoea ananatis: molecular oxygen, as it does aerobically in vitro, or a membrane quinone as it can anaerobically? The choice bears on how carotenogenesis is coupled to the cell's respiratory state.
Suggested experts: Beyer P, Sandmann G
Q: On which leaflet of the cytoplasmic membrane does the peripheral, probably monotopic CrtI sit, and does it associate physically with the upstream phytoene synthase CrtB and downstream cyclase CrtY to channel the insoluble carotene intermediates rather than releasing them into the bilayer?
Suggested experts: Schaub P, Cavarelli J
Experiment: Reconstitute purified CrtI with phytoene-loaded liposomes and measure lycopene formation and oxygen consumption under controlled oxygen tensions, then repeat anaerobically with a panel of native Pantoea quinones as the only available acceptor, quantifying turnover for each. Complement in vivo by assaying carotenoid accumulation in the organism under aerobic versus micro-aerobic growth.
Hypothesis: In its native aerobic physiology CrtI passes electrons to molecular oxygen, but under oxygen limitation it switches to a membrane quinone, so carotenogenesis continues anaerobically.
Type: reconstitution with defined electron acceptors
Experiment: Determine the sidedness of membrane-bound CrtI by protease-accessibility and labelling assays on inverted and right-side-out vesicles, and test for complex formation with CrtB and CrtY by co-purification, crosslinking and fluorescence microscopy of tagged functional alleles, comparing intermediate accumulation between strains expressing the enzymes together versus singly.
Hypothesis: CrtI is recruited monotopically to the cytoplasmic face of the inner membrane and forms a physical or kinetically coupled assembly with CrtB and CrtY that channels phytoene and lycopene.
Type: membrane topology and pathway-assembly mapping
Experiment: Assay purified CrtI against prolycopene under the anaerobic reduced-FAD conditions that permit isomerization and identify the products by HPLC and mass spectrometry, testing whether the enzyme reaches all-trans-lycopene or stops at partially isomerized tri-cis and di-cis species, and compare with an authentic CRTISO run in parallel.
Hypothesis: The physiological central-bond cis-to-trans isomerization is an obligatory internal step of the four-desaturation reaction rather than a separable carotenoid isomerase activity, and CrtI cannot fully convert prolycopene to all-trans-lycopene as a bona fide CRTISO would.
Type: substrate and isomerization dissection
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)