X-ray structure analysis of short-chain dehydrogenases/reductases

Sammanfattning: X-ray crystallography and site-directed mutagenesis were used to better understand the structure/function relationships in the family of short-chain dehydrogenases/reductases (SDR). This group of enzymes constitutes a protein family with at least 60 members with highly diverse functions in pro- and eukaryotes. Two enzymes of this family were studied in this work: Drosophila lebanonensis alcohol dehydrogenase (DADH; EC 1.1.1.1) and Comamonas testosteroni 3[beta]/17[beta]-hydroxysteroid dehydrogenase ([beta]/17[beta]-HSDH; EC 1.1.1.51). Drosophila alcohol dehydrogenase is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols to aldehydes/ketones. The crystal structures of the apoform, the binary form (E·NAD+) and three ternary complexes (E·NAD+·acetone, E·NAD+·3-pentanone and E·NAD+·cyclohexanone) were solved at 1.9 Å, 2.4 Å, 2.2 Å, 1.4 Å and 1.6 Å resolution, respectively. DADH subunits show an [alpha]/[beta] single domain structure with a characteristic NAD(H) binding motif (Rossmann fold). The peptide chain of a subunit is folded into a central eight-stranded [beta]-sheet flanked on each side by three [alpha]-helices. DADH forms homodimers that have local twofold symmetry. Dimer association is dominated by a four-helix bundle motif as well as two C-terminal loops from each subunit. The structure of DADH undergoes a conformational change in order to bind the coenzyme. Furthermore, upon binding of the ketone, a region that was disordered in the apoform (186-191) gets stabilized and closes the active site cavity by creating either a small helix (E·NAD+·acetone, E·NAD+·3-pentanone) or an ordered loop (E·NAD+·cyclohexanone). The active site pocket comprises a hydrophobic and bifurcated cavity that explains why the enzyme is more efficient in oxidizing secondary aliphatic alcohols -preferably R enantiomers- than primary ones. Difference Fourier maps showed that the ketone inhibitor molecule has undergone a covalent reaction with the coenzyme in all three ternary complexes. Due to the presence of the positively charged ring of the coenzyme (NAD+) and the residue Lys155, the amino acid Tyrl51 is in its deprotonated state at physiological pH. Tyr151 can abstract a proton from the enolic form of the ketone and catalyze a nucleophilic attack of the C[alpha] atom to the C4 position of the coenzyme creating an NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accounts for the inactivation of the enzyme. The alcohol oxidation proceeds in a similar way, involving the same amino acids as in the formation of the NAD-ketone adduct. Tyr151 abstracts a proton from the alcohol and therefore facilitates hydride transfer to the coenzyme. Ser138 has been studied by site-directed mutagenesis and its role is to stabilize the substrate/reaction transition state. Molecular models for the three alloenzymes of D.melanogaster ADH (ADH-S, -F and -UF) were built using the available crystal structure of D.lebanonensis ADH as a template. A model based on the analysis of the electrostatic potential distribution is presented to explain their different biochemical behavior. 3[beta]/17[beta]-hydroxysteroid dehydrogenase is one of the inducible enzymes produced by the gram-negative bacterium Comamonas testosteroni expressed upon adaptive growth in testosterone-containing media. Crystallization, preliminary X-ray analysis, plausible crystal packing and initial refinement procedures are shown. Mutagenesis work has been carried out in order to understand the structural basis of hormone metabolism and to give insight into the structure/function relationship in short-chain dehydrogenases/reductases.