Preparation of carotenoid cleavage dioxygenases for X-ray crystallography

Abstract

Carotenoid cleavage dioxygenases (CCDs) constitute a superfamily of enzymes that are found in all domains of life where they play key roles in the metabolism of carotenoids and apocarotenoids as well as certain phenylpropanoids such as resveratrol. Interest in these enzymes stems not only from their biological importance but also from their remarkable catalytic properties including their regioselectivity, their ability to accommodate diverse substrates, and the additional activities (e.g., isomerase) that some of these enzyme possess. X-ray crystallography is a key experimental approach that has allowed detailed investigation into the structural basis behind the interesting biochemical features of these enzymes. Here, we describe approaches used by our lab that have proven successful in generating single crystals of these enzymes in resting or ligand-bound states for high-resolution X-ray diffraction analysis.

Keywords: Apocarotenoid; Carotenoid; Cleavage dioxygenase; Cobalt; Crystallization; Enzyme; Metalloenzyme; Non-heme iron; X-ray crystallography.

Figure 1.. A CCD phylogeny and representative crystal structures from the different subfamilies.

The Bayesian phylogeny shows major groups of CCDs from plants (light green triangles), cyanobacteria (orange triangle), metazoans (yellow triangles), and the mixed bacterial/fungal SCO group (olive triangle). Also shown in purple text is an archaeal CCD from Nitrosotalea devanaterra, NdCCD. Groups for which at least one crystal structure has been determined are shown in bold. The crystal structures shown are as follows with PDB accession codes given in parentheses: bovine RPE65 (4F2Z), SynACO (4OU9), Neurospora crassa CAO1 (5U8X), Zea mays VP14 (3NPE), and NdCCD (6VCF).

Figure 2.. An overview of protocol described in this work for the production of diffraction-quality CCD crystals.

Each step is described in detail in the main text.

Figure 3.. Purification of SynACO from E. coli supernatant by ammonium sulfate fractionation and size-exclusion chromatography.

A) Coomassie-stained SDS-PAGE gel showing the step-wise precipitation of SynACO (band indicated by a white arrow) with increasing concentrations of ammonium sulfate. B) Chromatogram showing the elution of SynACO obtained from the 40% saturation pellet in panel A from a 120 mL Superdex 200 size-exclusion column. The peak indicated by arrow represents monomeric SynACO while the large peak at the front of the column is aggregated material. The inset shows a Coomassie-stained SDS-PAGE gel of the pooled fractions from size-exclusion chromatography. This figure is reused with permission from (Sui, Kiser, Che et al., 2014).

Figure 4.. Purification of NdCCD from E. coli supernatant by ion exchange and size-exclusion chromatography.

The left-most gel shows the starting supernatant (SN) loaded onto the High-trap Q HP column, the flow-through (FT), and fractions eluted by a linear gradient of NaCl. The NdCCD band migrates just above the 50 kDa molecular weight marker. The fractions shown were pooled, concentrated, and applied to a 120 mL Superdex 200 column. Eluted fractions from this column are shown in the middle gel. The final concentrated sample is shown in the rightmost gel. A minor impurity is seen just above the 25 kDa molecular weight marker. This figure is reused with permission from (Daruwalla, Zhang, Lee et al., 2020).

Figure 5.. The chemical basis of the TPTZ assay for ferrous iron.

Two molecules of TPTZ react with divalent iron to form a 6-coordinate complex with an optical absorbance band at ~596 nm. Any ferric iron in the sample can be converted to Fe²⁺ by addition of hydroxylamine to the assay system.

Figure 6.

CCD crystals obtained using the protocol described in this work. A) Obelisk-shaped orthorhombic SynACO crystals in space group P2₁2₁2₁. B) Trigonal CAO1 crystals in space group P3₁21. C) Hexagonal NdCCD crystals in space group P6₁. D) NdCCD crystals in the same form shown in panel C, but grown from protein purified in the presence of apocarotenoid substrate. Note their marked orange color. The scale bars in each panel represent approximately 100 μm. Panel D is reused with permission from (Daruwalla, Zhang, Lee et al., 2020)