HPLC co-chromatography, chemical modification and mass
spectrometry all identified compound 3 as 4-keto-a-carotene. This
is a unique carotenoid not reported thus far (Britton et al., 2004). A
similar but different carotenoid is 2-keto-a-carotene from the stick
insect Ectatosoma tiaratum (Kayser, 1981). Due to the position of
the keto group at C-2, it does not contribute to the conjugated
polyene chain which influences the optical absorbance spectrum
with three pronounced peaks at 423, 446 and 475 nm, distinct
from the spectrum in Fig. 3E. Formation of 4-keto-a-carotene is
due to the relatively high a-carotene content in the transgenic rice
callus. Obviously, the carotene ketolase from Brevundimonas sp.
possesses broad substrate specificity. This enzyme typically ketolates
both b-ionone rings in b-carotene (Nishida et al., 2005). In
addition, it is also able to ketolate the b-ionone end of the a-carotene
molecule. This broad substrate specificity in combination with
the a-carotene concentrations as substrate for the ketolase is the
primary reason for the formation of 4-keto-a-carotene in the
genetically engineered rice callus.