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Characterization of lycopene cyclase (CrtY) from marine bacterium, Paracoccus haeundaensis
Author(s) -
Kim Young Tae,
Jeong Tae Hyug,
Seo Yong Bae
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.499.3
Subject(s) - lycopene , chemistry , enzyme , cofactor , biochemistry , affinity chromatography , cyclase , carotenoid , stereochemistry
Lycopene cyclase (CrtY) converts lycopene into β‐carotene by catalyzing the formation of two beta‐rings at each end of the linear carotene. This reaction takes place as a two step reaction which both sides of the lycopene molecule are cyclized into β‐carotene rings via the monocyclic γ‐carotene as an intermediate. Lycopene cyclase gene ( crtY ) involved in the carotenoid biosynthetic pathway of the Paracoccus haeundaensis was subcloned into the expression vector, pET‐44a(+), and expressed in E. coli BL21(DE3) codon plus cell. The resulting protein was expressed as a fusion protein containing an N‐terminal six‐histidine extension which derived from the expression vector. The expressed protein was purified to homogeneity by affinity chromatography and incubated with the substrate mixture. The products of the enzymatic reaction were separated with HPLC and analyzed by spectroscopic method. The results showed that lycopene was converted into γ‐carotene and β‐carotene, suggesting that the purified enzyme (CrtY) is enzymatically active. Furthermore, substrate specificity and cofactor requirements of the purified protein were determined. The enzymatic reaction was stimulated by NADH or NADPH.