Gene Ontology annotation through association of InterPro records with GO terms

Supplied the GO:0016117 carotenoid biosynthetic process row from IPR014105 and the generic GO:0016491 oxidoreductase activity row from IPR002937, both correct for a CrtI-type FAD-dependent desaturase at family/domain level.

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

Mapped the Swiss-Prot Cell membrane subcellular location (SL-0039) to GO:0005886 plasma membrane, agreeing with the experimentally evidenced UniProt location.

GO_REF:0000117

Electronic Gene Ontology annotations created by ARBA machine learning models

Supplied electronic rows for GO:0016120 carotene biosynthetic process, GO:0016627 CH-CH-donor oxidoreductase activity and GO:0071949 FAD binding, each concordant with the direct experimental evidence for this enzyme.

PMID:1400305

Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase.

Purified the recombinant desaturase to homogeneity, reactivated it after urea solubilization, and showed directly that it converts 15-cis-phytoene to trans-lycopene, the source of the carotene-biosynthesis and CH-CH-oxidoreductase IDA rows.

"The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene."
Established FAD as the cofactor involved in desaturation and reported NAD and NADP as inhibitory, the source of the FAD-binding IDA row and of the UniProt activity-regulation record.

"FAD was involved in desaturation, whereas NAD and NADP were inhibitory."
Reported the first purification of a membrane-integrated carotenogenic enzyme in an active state, the experimental basis of the Cell membrane subcellular location.

"This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state."

PMID:2254247

Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli.

Cloned the six-gene carotenoid cluster of this organism and established that the single gene product CrtI performs the whole four-step conversion of phytoene to lycopene via phytofluene, zeta-carotene and neurosporene.

"only one gene product (CrtI) for the conversion of phytoene to lycopene is required, a conversion in which four sequential desaturations should occur via the intermediates phytofluene, zeta-carotene, and neurosporene."
Located the cluster on a defined genomic fragment from Erwinia uredovora 20D3 (ATCC 19321), the strain of the UniProt entry, fixing the gene identity.

"These genes were cloned from a phytopathogenic bacterium, Erwinia uredovora 20D3 (ATCC 19321), in Escherichia coli and located on a 6,918-bp fragment whose nucleotide sequence was determined."

PMID:22745782

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase.

Reconstituted the reaction in liposomes and showed clean conversion of 15-cis-phytoene to all-trans-lycopene with no build-up of intermediates, the transformation comprising four desaturations and one cis-trans isomerization.

"Right, CRTI-mediated phytoene desaturation encompassing all four desaturation steps and one cis-trans isomerization step to form all-trans-lycopene."
Concluded that FAD is the sole redox-active cofactor and that it is required structurally at membrane association, apoprotein being non-reactivatable.

"we conclude that FAD is the sole cofactor effective in CRTI-mediated phytoene desaturation"
Established that CrtI is a membrane-peripheral, probably monotopic oxidoreductase, revising the earlier membrane-integrated description while keeping the same cytoplasmic-membrane compartment.

"CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor."
Identified oxygen as the terminal electron acceptor aerobically, replaceable by quinones anaerobically, an open point for the native organism.

"Thus, oxygen must play the role of a terminal electron acceptor."
Showed that anaerobic isomerization of prolycopene by CRTI yields partially isomerized tri-cis and di-cis species rather than all-trans lycopene, distinguishing CrtI from a bona fide CRTISO carotenoid isomerase.

"led to the formation of a novel tri-cis-lycopene species accompanied by smaller amounts of the half-side isomerized 7,9-di-cis-lycopene"

PMID:39322757

Combining systems and synthetic biology for in vivo enzymology.

Quantified phytoene substrate and lycopene product intracellularly for Pantoea ananatis CrtI expressed in engineered yeast, corroborating the phytoene-in, lycopene-out activity of this specific protein in a living cell.

"For PaCrtI, the observed concentrations of phytoene (substrate) and lycopene (product) are either equivalent to or lower than those obtained with the two fungal CrtI enzymes."

file:PANAN/crtI/crtI-uniprot.txt

UniProt entry CRTI_PANAN (P21685)

Records the catalytic activity as 15-cis-phytoene + 4 A = all-trans-lycopene + 4 AH2 (Rhea:15585, EC 1.3.99.31), the overall four-step desaturation.

"Reaction=15-cis-phytoene + 4 A = all-trans-lycopene + 4 AH2;"
Records FAD as the cofactor and Cell membrane as the subcellular location, both with experimental evidence from PMID:1400305, and assigns the lycopene-biosynthesis pathway.

"Name=FAD; Xref=ChEBI:CHEBI:57692;"
Assigns the entry to the carotenoid/retinoid oxidoreductase family and matches the CrtI-specific NCBIfam model TIGR02734.

"DR NCBIfam; TIGR02734; crtI_fam; 1."