4c3y

Publication Abstract from PubMed

3-Ketosteroid Delta1-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 A resolution crystal structure of the 56 kDa enzyme from Rhodococcus erythropolis SQ1 (Delta1-KSTD1). The enzyme contains two domains, an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 A resolution structure of Delta1-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues, Tyr-119, Tyr-318, Tyr-487, and Gly-491. Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr-318, respectively, while the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr-487 and the backbone amide of Gly-491. Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism, in which Tyr-487 and Gly-491 work in tandem to promote keto-enol tautomerisation and increase the acidity of the substrate's C2 hydrogen atoms. With assistance of Tyr-119, the general base Tyr-318 abstracts the axial beta-hydrogen from C2 as a proton, while the FAD accepts the axial alpha-hydrogen from the substrate's C1 atom as a hydride ion.

Crystal structure and site-directed mutagenesis of 3-ketosteroid Delta1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism.,Rohman A, van Oosterwijk N, Thunnissen AM, Dijkstra BW J Biol Chem. 2013 Oct 28. PMID:24165124^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.