A 2.8 ANGSTROM RESOLUTION STRUCTURE OF 6-PHOSPHOGLUCONATE DEHYDROGENASE FROM THE PROTOZOAN PARASITE TRYPANOSOMA-BRUCEI - COMPARISON WITH THE SHEEP ENZYME ACCOUNTS FOR DIFFERENCES IN ACTIVITY WITH COENZYME AND SUBSTRATE-ANALOGS

The three-dimensional structure of 6-phosphogluconate dehydrogenase (6 PGDH) from the parasitic protozoan Trypanosoma brucci has been solved at 2.8 Angstrom resolution. This pentose phosphate pathway enzyme is N ADP-dependent; NADPH generated in the reaction protects against oxidat ive stress. The enzyme crystallises in the space-group P3(1)21 with a dimer in the asymmetric unit and cell dimensions a=b = 135.13 Angstrom , c=116.74 Angstrom, alpha=beta=90 degrees, gamma=120 degrees. The str ucture has refined to R=18.6% (R-free = 273%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that o f the sheep liver enzyme and significant activity differences have bee n observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate bin ding site. The tail of T. brucei 6PGDH is shorter than that of the she ep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates addition al interactions between the subunits close to the active site; the coe nzyme binding domain is thereby associated more tightly with the helic al domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the sub strate binding site. The differences could explain the 200-fold enhanc ed affinity observed for the substrate analogue 6-phospho-2-deoxy-D-gl uconate and suggest targets for anti-parasite drug design. The coenzym e binding domain of 6PGDH has a beta-alpha-beta fold; while in most sp ecies the ''fingerprint'' sequence is GxAxxG, in the T. brucei enzyme it is GxGxxC. Additional interactions between the enzyme and the coenz yme bis-phosphate are likely in the parasite 6PGDH, accounting for gre ater inhibition (40-fold) of 2'5'-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differ ences have been found between the monomers; those at the coenzyme bind ing site suggest the molecule could be asymmetric during the enzyme re action. (C) 1998 Academic Press.