Characteristics of a new carotenoid cleavage dioxygenase NtCCD10 derived from Nicotiana tabacum

Abstract

A new carotenoid cleavage dioxygenase NtCCD10 from tobacco was characterized. There is some difference between NtCCD10 and CCD1 in structure. NtCCD10 can cleave the C5-C6 (C5'-C6') and C9-C10 (C9'-C10') double bonds of carotenoids and has high catalytic activity. Carotenoid cleavage dioxygenases (CCDs) cleave carotenoids to produce a variety of apocarotenoids, which have important biological functions for organisms in nature. There are eleven CCDs subfamilies in the plant kingdom, many of which have been extensively characterized in their functions. However, as a newly classified subfamily, the function of CCD10 has rarely been studied. In this work, the function of an NtCCD10 gene from dicotyledonous Nicotiana tabacum was cloned and characterized, and its phylogeny, molecular structural modeling and protein structure were also systematically analyzed. Like other CCDs, NtCCD10 also possesses a seven bladed β-propeller with Fe²⁺ cofactor in its center constituting the active site of the enzyme. The Fe²⁺ is also coordinated bonding with four conserved histidine residues. Meanwhile, NtCCD10 also has many unique features, such as its α1 and α3 helixes are not anti-parallel, a special β-sheet and a longer access tunnel for substrates. When expressed in engineered Escherichia coli (producing phytoene, lycopene, β-carotene, and zeaxanthin) and Saccharomyces cerevisiae (producing β-carotene), NtCCD10 could symmetrically cleave phytoene and β-carotene at the C9-C10 and C9'-C10' positions to produce geranylacetone and β-ionone, respectively. In addition, NtCCD10 could also cleave the C5-C6 and C5'-C6' double bonds of lycopene to generate 6-methyl-5-heptene-2-one (MHO). NtCCD10 has higher catalytic activity than PhCCD1 in yeast, which provides a good candidate CCD for biosynthesis of β-ionone and has potential applications in biotechnological industry. This study identified the taxonomic position and catalytic activity of the first NtCCD10 in dicotyledonous plants. This will provide a reference for the discovery and functional identification of CCD10 enzymes in dicotyledons.

Keywords: Apocarotenoid; Carotenoid cleavage dioxygenase 10; Nicotiana tabacum; β-Ionone.