Z-ISO (15-cis-zeta-carotene isomerase, EC 5.2.1.12; At1g10830) is a nuclear-encoded, plastid-targeted integral membrane enzyme of the poly-cis branch of carotenoid biosynthesis found in plants, algae, diatoms and cyanobacteria. It catalyses the cis-to-trans isomerization of the central 15-cis (15-15') double bond of 9,9',15-tri-cis-zeta-carotene to give 9,9'-di-cis-zeta-carotene (Rhea:30967). In the plastid poly-cis desaturation route it acts between the two plastoquinone-dependent desaturases: phytoene desaturase (PDS) produces the tri-cis-zeta-carotene substrate, Z-ISO isomerizes its 15-cis bond, and zeta-carotene desaturase (ZDS) then converts the product toward prolycopene, which prolycopene isomerase (CRTISO) isomerises to all-trans-lycopene, the precursor of all cyclic carotenes and xanthophylls. Isomerization of the 15-cis bond can also occur photochemically in the light, so Z-ISO is strictly required for carotenoid formation in the dark and in poorly illuminated plastids (endosperm, roots, etiolated seedlings) and enhances pathway efficiency in illuminated tissue; loss of function abolishes carotenoids in the dark and delays greening. The protein carries an N-terminal chloroplast transit peptide and multiple transmembrane helices, resides in plastid (chloroplast) membranes, and is an unusual heme-b-dependent isomerase whose activity requires the reduced (ferrous) heme cofactor. Its NnrU domain (Pfam PF07298) reflects an evolutionary origin from a bacterial NnrU-related membrane protein recruited into carotenogenesis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0009507 chloroplast | HDA PMID:18431481 Sorting signals, N-terminal modifications and abundance of t... | ACCEPT | Summary: High-throughput chloroplast-proteome detection (van Wijk group) placing Z-ISO in the chloroplast. Consistent with the experimentally supported N-terminal chloroplast transit peptide, with UniProt's plastid membrane assignment, and with the strictly plastidial carotenoid pathway in which the enzyme acts. The cached full text does not name At1g10830 in its body (the gene-level datum is in the large-scale proteomics supplementary data), so the location is supported here from the UniProt record. Reason: Correct organelle for a plastid carotenoid biosynthetic enzyme whose precursor carries a chloroplast transit peptide and whose substrate and product are membrane-embedded plastid carotenes. Retained as a core location (the more specific site is the chloroplast membrane, below). Supporting Evidence: file:ARATH/Z-ISO/Z-ISO-uniprot.txt SUBCELLULAR LOCATION: Plastid, chloroplast membrane {ECO:0000305}; |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | ACCEPT | Summary: Sequence-based (AtSubP) prediction of chloroplast localization; a duplicate of the same correct compartment established by proteomics and by the transit peptide. Reason: Consistent with the HDA chloroplast annotation, the transit peptide and the UniProt plastid assignment. Duplicate location annotations with a different evidence code are acceptable. Supporting Evidence: file:ARATH/Z-ISO/Z-ISO-uniprot.txt SUBCELLULAR LOCATION: Plastid, chloroplast membrane {ECO:0000305}; |
| GO:0016120 carotene biosynthetic process | IMP PMID:20335404 Isolation and characterization of the Z-ISO gene encoding a ... | ACCEPT | Summary: Chen et al. isolated maize and Arabidopsis Z-ISO mutants and identified the gene, showing that the enzyme performs the 15-cis isomerization step required to convert 15-cis-phytoene toward all-trans-lycopene; the mutant lacks carotenoids in the dark. Z-ISO's substrate and product are both zeta-carotenes (carotenes), and it catalyses a step of the carotene backbone route, so carotene biosynthetic process is the correct process. Cached record is abstract-only (full_text_available: false) but states the functional and phenotypic result explicitly. Reason: Core biological process: Z-ISO itself performs a catalytic step of carotene biosynthesis, not merely a requirement for it. Curator use of the acts_upstream_of_or_within qualifier does not weaken the term choice, which is well matched to the enzyme's product (a carotene). Supporting Evidence: PMID:20335404 Functional testing of the gene product in Escherichia coli showed isomerization of the 15-cis double bond in 9,15,9'-tri-cis-zeta-carotene, proving that Z-ISO encoded the missing step. PMID:20335404 Z-ISO was found to be important for both light-exposed and "dark" tissues. |
| GO:0031969 chloroplast membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Mapped from the UniProt subcellular location 'Plastid, chloroplast membrane; Multi-pass membrane protein'. Membrane residence is strongly supported: UniProt predicts six transmembrane helices, and biochemical reconstitution of plant Z-ISO showed it is an integral membrane protein that acts on membrane-embedded carotene substrates. GO:0031969 denotes the plastid envelope/thylakoid bilayers; the precise sub-plastid membrane for Z-ISO is not resolved. Reason: Consistent with UniProt, the predicted transmembrane topology, and the demonstrated integral-membrane character of the enzyme. Accepted as the core site of action while noting that the envelope-versus-thylakoid assignment is not established. Supporting Evidence: file:ARATH/Z-ISO/Z-ISO-uniprot.txt Multi-pass membrane protein {ECO:0000305}. PMID:26075523 Z-ISO is a bona fide enzyme and integral membrane protein. |
| GO:0090471 9,15,9'-tri-cis-zeta-carotene isomerase activity | IDA PMID:20335404 Isolation and characterization of the Z-ISO gene encoding a ... | ACCEPT | Summary: Direct assay: Z-ISO expressed in E. coli isomerized the 15-cis double bond of 9,15,9'-tri-cis-zeta-carotene, the defining reaction of the enzyme (EC 5.2.1.12, Rhea:30967). This is the core molecular function. Later biochemistry on the maize ortholog showed the enzyme independently catalyses the 15-15' cis-trans isomerization using a ferrous heme b cofactor. Reason: The experimentally established core activity of the protein, at exactly the right level of specificity. GO:0090471 corresponds to EC 5.2.1.12 and Rhea:30967, matching the reaction demonstrated in Arabidopsis and maize Z-ISO. Supporting Evidence: PMID:20335404 Functional testing of the gene product in Escherichia coli showed isomerization of the 15-cis double bond in 9,15,9'-tri-cis-zeta-carotene, proving that Z-ISO encoded the missing step. PMID:26075523 Z-ISO independently catalyzes the cis-trans isomerization of the 15-15' carbon-carbon double bond in |
| GO:0090471 9,15,9'-tri-cis-zeta-carotene isomerase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated annotation from the EC 5.2.1.12 / Rhea:30967 mapping to the same isomerase activity. The mapped reaction is exactly the one demonstrated experimentally for this protein. Reason: Correct and appropriately specific; the electronic EC/Rhea mapping coincides with the experimental IDA row. Duplicate function annotations with different evidence codes are acceptable. Supporting Evidence: file:ARATH/Z-ISO/Z-ISO-uniprot.txt Reaction=9,9',15-tri-cis-zeta-carotene = 9,9'-di-cis-zeta-carotene; file:ARATH/Z-ISO/Z-ISO-uniprot.txt EC=5.2.1.12; |
| GO:0016117 carotenoid biosynthetic process | IDA PMID:20335404 Isolation and characterization of the Z-ISO gene encoding a ... | NEW | Summary: Proposed process annotation at the carotenoid-pathway level. Z-ISO catalyses a committed step of the route that builds carotenoids, and its loss abolishes carotenoids in the dark. Reason: Participation test passed: Z-ISO itself performs a catalytic step of carotenoid biosynthesis. Comparator check passed: the sibling pathway enzyme CRTISO carries GO:0016117 and the carotene backbone module uses GO:0016117 as the module-level process. Not redundant with the existing GO:0016120 carotene biosynthetic process: in the current ontology GO:0016120 is not a descendant of GO:0016117, so a GO:0016117 query otherwise under-retrieves Z-ISO. Added conservatively to place the enzyme in the standard carotenoid-biosynthesis retrieval branch. Supporting Evidence: PMID:20335404 Functional testing of the gene product in Escherichia coli showed isomerization of the 15-cis double bond in 9,15,9'-tri-cis-zeta-carotene, proving that Z-ISO encoded the missing step. file:ARATH/Z-ISO/Z-ISO-uniprot.txt FUNCTION: Isomerase involved in the biosynthesis of carotenoids. |
| GO:0020037 heme binding | ISS PMID:26075523 Control of carotenoid biosynthesis through a heme-based cis-... | NEW | Summary: Molecular function not present in GOA but supported by biochemistry: plant Z-ISO requires a ferrous heme b cofactor with redox-regulated axial-ligand switching for catalysis. The decisive purification and spectroscopy used the maize ortholog, so the annotation for Arabidopsis Q9SAC0 is by orthology/sequence similarity. Reason: Heme binding is a direct molecular function of the protein (a cofactor it binds and uses catalytically), demonstrated for plant Z-ISO and conserved across the Z-ISO clade. Proposed conservatively as ISS because the experimental evidence is on the maize ortholog rather than on Arabidopsis Q9SAC0 itself. Supporting Evidence: PMID:26075523 isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. |
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Download this section (compressed HTML)Q: Does Arabidopsis Z-ISO (Q9SAC0) itself, rather than the maize ortholog, bind a ferrous heme b cofactor and use the same redox-regulated axial-ligand-switching mechanism, and which residues (e.g. the candidate histidines) provide the axial ligands?
Suggested experts: Eleanore T. Wurtzel
Q: In which plastid membrane (inner envelope versus thylakoid) does Arabidopsis Z-ISO reside, and does it associate physically with PDS/ZDS in a desaturation complex? The current 'chloroplast membrane' annotation rests on UniProt similarity and proteomics rather than direct sub-plastid localization of the Arabidopsis protein.
Suggested experts: Barry J. Pogson
Q: What is the in vivo electron/redox donor that keeps the Z-ISO heme in the active ferrous state within the plastid membrane, and how is this coordinated with the plastoquinone pool used by the flanking desaturases?
Suggested experts: Eleanore T. Wurtzel
Experiment: Express and purify recombinant mature Arabidopsis Z-ISO (residues 59-367) as a cleavable MBP fusion, confirm bound heme b by pyridine-hemochrome and UV-visible/EPR spectroscopy, and assay isomerization in phosphatidylcholine liposomes loaded with purified 9,9',15-tri-cis-zeta-carotene under reducing (dithionite) versus oxidizing conditions; follow substrate-to-product conversion by C30 reversed-phase HPLC with photodiode-array detection.
Hypothesis: Arabidopsis Z-ISO is a ferrous-heme-b-dependent integral-membrane isomerase that converts 9,9',15-tri-cis-zeta-carotene to 9,9'-di-cis-zeta-carotene.
Type: in vitro enzyme assay with purified recombinant protein
Experiment: Combine native-promoter Z-ISO-GFP complementation of an Arabidopsis z-iso mutant (checking rescue of the dark carotenoid phenotype) with immunoblotting of purified envelope, stroma and thylakoid fractions and carbonate/salt-wash tests of membrane association; probe PDS/ZDS co-migration by blue-native PAGE.
Hypothesis: Z-ISO is a peripheral or integral protein of a specific plastid membrane and is co-located with the poly-cis desaturases.
Type: subcellular localization by fluorescence microscopy and fractionation
Experiment: Complement an Arabidopsis z-iso null and a cyanobacterial z-iso deletion with plant Z-ISO versus a bacterial NnrU protein, scoring restoration of di-cis-zeta-carotene formation and downstream carotenoids; test structure- guided heme-ligand mutants for loss of activity.
Hypothesis: The NnrU ancestry of Z-ISO is not sufficient for carotenoid-isomerase activity; the acquired heme-based isomerase function is lineage-specific.
Type: cross-complementation with structure-guided mutagenesis
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