Publications

• Characterization of bacterial beta-carotene 3,3'-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli.

  Choi S.K., Matsuda S., Hoshino T., Peng X., Misawa N.

  beta-Carotene hydroxylase (CrtZ) is one of rate-limiting enzymes for astaxanthin production. A complementation analysis was conducted using Escherichia coli transformants to compare the catalytic efficiency of bacterial CrtZ from Brevundimonas sp. SD212, Paracoccus sp. PC1 (formerly known as Alcal ... >> More

  beta-Carotene hydroxylase (CrtZ) is one of rate-limiting enzymes for astaxanthin production. A complementation analysis was conducted using Escherichia coli transformants to compare the catalytic efficiency of bacterial CrtZ from Brevundimonas sp. SD212, Paracoccus sp. PC1 (formerly known as Alcaligenes sp. PC-1), Paracoccus sp. N81106 (Agrobacterium aurantiacum), Pantoea ananatis (Erwinia uredovora 20D3), marine bacterium P99-3, and P450 monooxygenase (CYP175A1) from Thermus thermophilus HB27. Each crtZ or CYP175A1 gene was expressed in E. coli transformants synthesizing canthaxanthin and beta-carotene due to the respective presence of plasmids pAC-Cantha and pACCAR16DeltacrtX. The carotenoids that accumulated in the resulting recombinant E. coli cells were examined by chromatographic and spectroscopic analyses. E. coli carrying Brevundimonas sp. SD212 crtZ showed the highest astaxanthin production efficiency among the transformants examined, while there was no significant difference in the catalytic efficiency for conversion from beta-carotene to zeaxanthin. Recombinant E. coli expressing the CYP175A1 gene, in addition to the genes for canthaxanthin synthesis, surprisingly accumulated adonirubin (phoenicoxanthin) as the main product, although the other recombinant E. coli did not accumulate any adonirubin. The present results suggest that the Brevundimonas sp. SD212 crtZ and T. thermophilus HB27 CYP175A1 genes could, respectively, be used for the efficient production of astaxanthin and adonirubin in heterologous hosts. << Less

  Appl Microbiol Biotechnol 72:1238-1246(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L.).

  Bouvier F., Keller Y., d'Harlingue A., Camara B.

  To dissect the mechanism by which carotenoid hydroxylases catalyze xanthophyll formation, we have cloned two pepper cDNAs encoding beta-cryptoxanthin and zeaxanthin biosynthetic enzymes. Using an in vitro system, we find that both enzymes are ferredoxin dependent and that their activity is strongl ... >> More

  To dissect the mechanism by which carotenoid hydroxylases catalyze xanthophyll formation, we have cloned two pepper cDNAs encoding beta-cryptoxanthin and zeaxanthin biosynthetic enzymes. Using an in vitro system, we find that both enzymes are ferredoxin dependent and that their activity is strongly inhibited by iron chelators such as o-phenanthroline or 8-hydroxyquinoline. This suggests the transfer of a reducing equivalent from NADPH to the hydroxylase via ferredoxin and the involvement of an iron activated oxygen insertion process. Based on sequence analysis, the putative histidine clusters involved in the iron coordination were identified and their roles evaluated. Following site-directed mutagenesis of the identified histidine residues hydroxylase activity was totally inactivated. Collectively, our data indicate that carotenoid hydroxylases belong to a new class of diiron proteins structurally related to membrane fatty acid desaturases. Mechanistically, both types of enzymes exploit iron activated oxygen to break the C-H bond with concomitant formation of double bond or oxygen insertion. We propose that the same mechanism operates for beta-carotene ketolase and probably for other carotenoid oxygenases as well. << Less

  Biochim. Biophys. Acta 1391:320-328(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation.

  Linden H.

  A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum ... >> More

  A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis. << Less

  Biochim. Biophys. Acta 1446:203-212(1999) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development.

  Zhu C., Yamamura S., Nishihara M., Koiwa H., Sandmann G.

  cDNAs encoding lycopene epsilon -cyclase, lycopene beta-cyclase, beta-carotene hydroxylase and zeaxanthin epoxidase were isolated from a Gentiana lutea petal cDNA library. The function of all cDNAs was analyzed by complementation in Escherichia coli. Transcript levels during different stages of fl ... >> More

  cDNAs encoding lycopene epsilon -cyclase, lycopene beta-cyclase, beta-carotene hydroxylase and zeaxanthin epoxidase were isolated from a Gentiana lutea petal cDNA library. The function of all cDNAs was analyzed by complementation in Escherichia coli. Transcript levels during different stages of flower development of G. lutea were determined and compared to the carotenoid composition. Expression of all genes increased by a factor of up to 2, with the exception of the lycopene epsilon -cyclase gene. The transcript amount of the latter was strongly decreased. These results indicate that during flower development, carotenoid formation is enhanced. Moreover, metabolites are shifted away from the biosynthetic branch to lutein and are channeled into beta-carotene and derivatives. << Less

  Biochim. Biophys. Acta 1625:305-308(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Cloning and functional analysis of the beta-carotene hydroxylase of Arabidopsis thaliana.

  Sun Z., Gantt E., Cunningham F.X. Jr.

  An Arabidopsis thaliana cDNA encoding the enzyme beta-carotene hydroxylase was identified by functional complementation in Escherichia coli. The product of this cDNA adds hydroxyl groups to both beta rings of the symmetrical beta-carotene (beta,beta-carotene) to form zeaxanthin (beta,beta-carotene ... >> More

  An Arabidopsis thaliana cDNA encoding the enzyme beta-carotene hydroxylase was identified by functional complementation in Escherichia coli. The product of this cDNA adds hydroxyl groups to both beta rings of the symmetrical beta-carotene (beta,beta-carotene) to form zeaxanthin (beta,beta-carotene-3,3'-diol) and converts the monocyclic beta-zeacarotene (7',8'-dihydro-beta,psi-carotene) to hydroxy-beta-zeacarotene (7',8'-dihydro-beta,psi-carotene-3-ol). The epsilon rings of delta-carotene (epsilon,psi-carotene) and alpha-zeacarotene (7',8'-dihydro-epsilon,psi-carotene) are poor substrates for the enzyme. The predicted amino acid sequence of the A. thaliana enzyme resembles the four known bacterial beta-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids. Truncation of the cDNA to produce a polypeptide lacking the first 69 amino acids does not impair enzyme activity in E. coli. Truncation to yield a polypeptide of a length comparable with the bacterial enzymes (lacking 129 N-terminal amino acids) resulted in the accumulation of the monohydroxy intermediate beta-cryptoxanthin (beta,beta-carotene-3-ol), predominantly, when beta-carotene was provided as the substrate. It is suggested that amino acid residues 70-129 of the A. thaliana enzyme may play a role in formation of a functional homodimer. << Less

  J. Biol. Chem. 271:24349-24352(1996) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• In vitro characterization of astaxanthin biosynthetic enzymes.

  Fraser P.D., Miura Y., Misawa N.

  Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haematococcus pluvialis bkt, and Erwinia uredovora crtZ genes were used for in vitro characterization of the respective enzymes. Specific ... >> More

  Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haematococcus pluvialis bkt, and Erwinia uredovora crtZ genes were used for in vitro characterization of the respective enzymes. Specific enzyme assays indicated that all of the enzymes are bifunctional, in that the CrtZ enzymes formed zeaxanthin from beta-carotene via beta-cryptoxanthin, as well as astaxanthin from canthaxanthin via phoenicoxanthin (adonirubin). The BKT/CrtW enzymes synthesized canthaxanthin via echinenone from beta-carotene and 4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from zeaxanthin. Comparison of maximum catalytic activities as well as selectivity experiments carried out in the presence of both utilizable substrates indicated that the CrtZ enzymes from marine bacteria converted canthaxanthin to astaxanthin preferentially, whereas the Erwinia CrtZ possessed a favorability to the formation of zeaxanthin from beta-carotene. The CrtW/BKT enzymes were not so defined in their substrate preference, responding readily to fluctuations in substrate levels. Other properties obtained indicated that the enzymes were strictly oxygen-requiring; and a cofactor mixture of 2-oxoglutarate, ascorbic acid, and Fe2+ was beneficial to activity. Based on enzymological data, a predicted pathway for astaxanthin biosynthesis is described, and it is proposed that CrtZ-like enzymes be termed carotenoid 3, (3')-beta-ionone ring hydroxylase and CrtW/BKT carotenoid 4, (4')-beta-ionone ring oxygenase. << Less

  J Biol Chem 272:6128-6135(1997) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay.

  Fraser P.D., Shimada H., Misawa N.

  An in vitro assay procedure for the carotenoid (beta-ionone ring) 3,3'-hydroxylase and 4,4'-oxygenase has been developed that enables efficient conversion of non-radiolabeled carotenoid substrates added directly into aqueous solution. The following enzymic conversions were demonstrated and apparen ... >> More

  An in vitro assay procedure for the carotenoid (beta-ionone ring) 3,3'-hydroxylase and 4,4'-oxygenase has been developed that enables efficient conversion of non-radiolabeled carotenoid substrates added directly into aqueous solution. The following enzymic conversions were demonstrated and apparent kinetic constants (Vmax, Km, and specificity constants) obtained: (a) 3,3'-hydroxylase (from Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) converted phoenicoxanthin (adonirubin) to astaxanthin, 3-hydroxyechinenone to 4-ketozeaxanthin (adonixanthin), 3'-hydroxyechinenone to 4-ketozeaxanthin, as well as echinenone to 4-ketozeaxanthin via 3- and 3'-hydroxyechinenone; (b) 4,4'-Oxygenase (from A. aurantiacum, Alcaligenes sp. strain PC-1 and Haematococcus pluvialis) converted 4-ketozeaxanthin to astaxanthin, 3-hydroxyechinenone to phoenicoxanthin, 3'-hydroxyechinenone to phoenicoxanthin, and echinenone to canthaxanthin. Determination of substrate specifities allowed assessment of biosynthetic routes to astaxanthin formation and demonstrated that pathways via mono-hydroxylated and ketolated products are enzymically feasible. << Less

  Eur J Biochem 252:229-236(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.