Preparation of carotenoid cleavage dioxygenases for X-ray crystallography.

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

Preparation and characterization of metal-substituted carotenoid cleavage oxygenases.

Carotenoid cleavage oxygenases (CCO) are non-heme iron enzymes that catalyze oxidative cleavage of alkene bonds in carotenoid and stilbenoid substrates. Previously, we showed that the iron cofactor of CAO1, a resveratrol-cleaving member of this family, can be substituted with cobalt to yield a catalytically inert enzyme useful for trapping active site-bound stilbenoid substrates for structural characterization. Metal substitution may provide a general method for identifying the natural substrates for CCOs in addition to facilitating structural and biophysical characterization of CCO-carotenoid complexes under normal aerobic conditions. Here, we demonstrate the general applicability of cobalt substitution in a prototypical carotenoid cleaving CCO, apocarotenoid oxygenase (ACO) from Synechocystis. Among the non-native divalent metals investigated, cobalt was uniquely able to stably occupy the ACO metal binding site and inhibit catalysis. Analysis by X-ray crystallography and X-ray absorption spectroscopy demonstrate that the Co(II) forms of both ACO and CAO1 exhibit a close structural correspondence to the native Fe(II) enzyme forms. Hence, cobalt substitution is an effective strategy for generating catalytically inert but structurally intact forms of CCOs

Analysis of Carotenoid Isomerase Activity in a Prototypical Carotenoid Cleavage Enzyme, Apocarotenoid Oxygenase (ACO)*

Carotenoid cleavage enzymes (CCEs) constitute a group of evolutionarily related proteins that metabolize a variety of carotenoid and non-carotenoid substrates. Typically, these enzymes utilize a non-heme iron center to oxidatively cleave a carbon-carbon double bond of a carotenoid substrate. Some members also isomerize specific double bonds in their substrates to yield cis-apocarotenoid products. The apocarotenoid oxygenase from Synechocystis has been hypothesized to represent one such member of this latter category of CCEs. Here, we developed a novel expression and purification protocol that enabled production of soluble, native ACO in quantities sufficient for high resolution structural and spectroscopic investigation of its catalytic mechanism. High performance liquid chromatography and Raman spectroscopy revealed that ACO exclusively formed all-trans products. We also found that linear polyoxyethylene detergents previously used for ACO crystallization strongly inhibited the apocarotenoid oxygenase activity of the enzyme. We crystallized the native enzyme in the absence of apocarotenoid substrate and found electron density in the active site that was similar in appearance to the density previously attributed to a di-cis-apocarotenoid intermediate. Our results clearly demonstrated that ACO is in fact a non-isomerizing member of the CCE family. These results indicate that careful selection of detergent is critical for the success of structural studies aimed at elucidating structures of CCE-carotenoid/retinoid complexes

Mechanism of action for small-molecule inhibitors of triacylglycerol synthesis

Abstract Inhibitors of triacylglycerol (TG) synthesis have been developed to treat metabolism-related diseases, but we know little about their mechanisms of action. Here, we report cryo-EM structures of the TG-synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a membrane bound O-acyltransferase (MBOAT), in complex with two different inhibitors, T863 and DGAT1IN1. Each inhibitor binds DGAT1’s fatty acyl-CoA substrate binding tunnel that opens to the cytoplasmic side of the ER. T863 blocks access to the tunnel entrance, whereas DGAT1IN1 extends further into the enzyme, with an amide group interacting with more deeply buried catalytic residues. A survey of DGAT1 inhibitors revealed that this amide group may serve as a common pharmacophore for inhibition of MBOATs. The inhibitors were minimally active against the related MBOAT acyl-CoA:cholesterol acyltransferase 1 (ACAT1), yet a single-residue mutation sensitized ACAT1 for inhibition. Collectively, our studies provide a structural foundation for developing DGAT1 and other MBOAT inhibitors

The structure of phosphatidylinositol remodeling MBOAT7 reveals its catalytic mechanism and enables inhibitor identification

Cells remodel glycerophospholipid acyl chains via the Lands cycle to adjust membrane properties. Membrane-bound O-acyltransferase (MBOAT) 7 acylates lyso-phosphatidylinositol (lyso-PI) with arachidonyl-CoA. MBOAT7 mutations cause brain developmental disorders, and reduced expression is linked to fatty liver disease. In contrast, increased MBOAT7 expression is linked to hepatocellular and renal cancers. The mechanistic basis of MBOAT7 catalysis and substrate selectivity are unknown. Here, we report the structure and a model for the catalytic mechanism of human MBOAT7. Arachidonyl-CoA and lyso-PI access the catalytic center through a twisted tunnel from the cytosol and lumenal sides, respectively. N-terminal residues on the ER lumenal side determine phospholipid headgroup selectivity: swapping them between MBOATs 1, 5, and 7 converts enzyme specificity for different lyso-phospholipids. Finally, the MBOAT7 structure and virtual screening enabled identification of small-molecule inhibitors that may serve as lead compounds for pharmacologic development

Utilization of Dioxygen by Carotenoid Cleavage Oxygenases

Carotenoid cleavage oxygenases (CCOs) are non-heme, Fe(II)-dependent enzymes that participate in biologically important metabolic pathways involving carotenoids and apocarotenoids, including retinoids, stilbenes, and related compounds. CCOs typically catalyze the cleavage of non-aromatic double bonds by dioxygen (O(2)) to form aldehyde or ketone products. Expressed only in vertebrates, the RPE65 sub-group of CCOs catalyzes a non-canonical reaction consisting of concerted ester cleavage and trans-cis isomerization of all-trans-retinyl esters. It remains unclear whether the former group of CCOs functions as mono- or di-oxygenases. Additionally, a potential role for O(2) in catalysis by the RPE65 group of CCOs has not been evaluated to date. Here, we investigated the pattern of oxygen incorporation into apocarotenoid products of Synechocystis apocarotenoid oxygenase. Reactions performed in the presence of (18)O-labeled water and (18)O(2) revealed an unambiguous dioxygenase pattern of O(2) incorporation into the reaction products. Substitution of Ala for Thr at position 136 of apocarotenoid oxygenase, a site predicted to govern the mono- versus dioxygenase tendency of CCOs, greatly reduced enzymatic activity without altering the dioxygenase labeling pattern. Reevaluation of the oxygen-labeling pattern of the resveratrol-cleaving CCO, NOV2, previously reported to be a monooxygenase, using a purified enzyme sample revealed that it too is a dioxygenase. We also demonstrated that bovine RPE65 is not dependent on O(2) for its cleavage/isomerase activity. In conjunction with prior research, the results of this study resolve key issues regarding the utilization of O(2) by CCOs and indicate that dioxygenase activity is a feature common among double bond-cleaving CCOs