STRUCTURE OF TRYPANOTHIONE REDUCTASE FROM CRITHIDIA-FASCICULATA AT 2.6 ANGSTROM RESOLUTION - ENZYME-NADP INTERACTIONS AT 2.8 ANGSTROM RESOLUTION

Trypanothione reductase is an FAD-dependent disulfide oxidoreductase w hich catalyses the reduction of trypanothione using NADPH as co-factor . The enzyme is unique to protozoan parasites from the genera Trypanos oma and Leishmania and is an important target for the design of improv ed anti-trypanocidal drugs. We present details of the structure of try panothione reductase from Crithidia fasciculata solved by molecular re placement, using human glutathione reductase as a search model, and re fined to an R factor of 16.1% with data between 8.0 and 2.6 angstrom r esolution. The model comprises two subunits (one containing 487 residu es, the other 486), an FAD prosthetic group, plus 392 solvent molecule s. The last four C-terminal residues are not seen in either subunit an d the density is poor for the N-terminal residue of subunit B. The mod el has a root-mean-square deviation from ideality of 0.016 angstrom fo r bond lengths and 3.2-degrees for bond angles. Each subunit was indep endently refined in the latter stages of the analysis but the subunits remain similar as indicated by the root-mean-square deviation of 0.35 angstrom for C(alpha) atoms. Trypanothione reductase has 36% sequence identity with human glutathione reductase and the root-mean-square de viation between the 462 C(alpha) atoms in the secondary structural uni ts common to the two proteins is 1.1 angstrom. However, there are larg e differences in the loop regions and significant shifts in the orient ation of the four domains within each subunit. Domain II, which binds the dinucleotide co-factor, and domain IV, which forms the interface b etween the two subunits, are both rotated by approximately 5-degrees w ith respect to domain I, which binds the FAD moiety, when compared wit h glutathione reductase. Crystals of trypanothione reductase have been soaked in the dinucleotide co-factor NADPH and N1-glutathionyl-spermi dine disulfide substrate and the structure of the resulting complex de termined at 2.8 angstrom resolution. Strong density is observed for th e adenosine end of the co-factor which forms many charged interactions with the protein though the density for the nicotinamide moiety is mo re diffuse. The mode of binding indicates that NADP is bound to the en zyme in a similar conformation to that observed with human glutathione reductase.