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A 2.8 A 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 analogues.
J Mol Biol. 1998 Sep 25; 282(3):667-81.JM

Abstract

The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 A resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P3121 with a dimer in the asymmetric unit and cell dimensions a=b=135.13 A, c=116.74 A, alpha=beta=90 degrees, gamma=120 degrees. The structure has refined to R=18.6% (Rfree=27.3%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy-D-gluconate and suggest targets for anti-parasite drug design. The coenzyme binding domain of 6PGDH has a beta-alpha-beta fold; while in most species the "fingerprint" sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2'5'-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction.

Authors+Show Affiliations

Department of Biochemistry, Oxford University, Rex Richards Building, Oxford, OX1 3QU, UK.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9737929

Citation

Phillips, C, et al. "A 2.8 a 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 Analogues." Journal of Molecular Biology, vol. 282, no. 3, 1998, pp. 667-81.
Phillips C, Dohnalek J, Gover S, et al. A 2.8 A 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 analogues. J Mol Biol. 1998;282(3):667-81.
Phillips, C., Dohnalek, J., Gover, S., Barrett, M. P., & Adams, M. J. (1998). A 2.8 A 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 analogues. Journal of Molecular Biology, 282(3), 667-81.
Phillips C, et al. A 2.8 a 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 Analogues. J Mol Biol. 1998 Sep 25;282(3):667-81. PubMed PMID: 9737929.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A 2.8 A 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 analogues. AU - Phillips,C, AU - Dohnalek,J, AU - Gover,S, AU - Barrett,M P, AU - Adams,M J, PY - 1998/9/17/pubmed PY - 1998/9/17/medline PY - 1998/9/17/entrez SP - 667 EP - 81 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 282 IS - 3 N2 - The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 A resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P3121 with a dimer in the asymmetric unit and cell dimensions a=b=135.13 A, c=116.74 A, alpha=beta=90 degrees, gamma=120 degrees. The structure has refined to R=18.6% (Rfree=27.3%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy-D-gluconate and suggest targets for anti-parasite drug design. The coenzyme binding domain of 6PGDH has a beta-alpha-beta fold; while in most species the "fingerprint" sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2'5'-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/9737929/A_2_8_A_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_analogues_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(98)92059-9 DB - PRIME DP - Unbound Medicine ER -