Stearoyl-acyl carrier protein desaturase (SAD) is a diiron cluster-containing enzyme that catalyzes the specific introduction of double bonds into fatty acyl chains. The core of SAD, consisting of four helices, is structurally similar to other diiron-containing enzymes such as peroxidase, rubrerythrin, and methane monooxygenase (MMO). Our previous study of SAD in Ricinus communis mapped the contribution of specific mutated residues to the change in product specificity from desaturation to hydroxylation. These mutations in the SAD protein were based on the MMO protein, for which the hydroxylation reaction mechanism is described. Ricinus communis SAD is a soluble enzyme, and its structure is known even in complex with the acyl carrier protein. However, the orientation of fatty acyls in the active site of the SAD, the impact of fatty acids on protein structure, and the precise desaturation reaction mechanism remain elusive.
In this study we have co-crystallized SAD from Ricinus communis with modified substrates, to obtain a protein-ligand complex that reveals the geometry of the substrate in the active site. To obtain crystals of a protein-ligand complex, the stearoyl-CoA substrate was modified by the addition of an oxime or keto group with chelating properties. Co-crystallization with the modified substrates led to the successful determination of the 3D protein-ligand structure using X-ray crystallography. The newly obtained structure represents the first soluble SAD structure with electron density for the fatty acyl chain in the protein structure. This crystallographic data will be used for the precise simulation of the reaction mechanism using the QM/MM approach, thus contributing to the knowledge of the reaction mechanism.