Berta M. Martins

NAD⁺‐dependent (R)‐2‐hydroxyglutarate dehydrogenase (HGDH) catalyses the reduction of 2‐oxoglutarate to (R)‐2‐hydroxyglutarate and belongs to the d‐2‐hydroxyacid NAD⁺‐dependent dehydrogenase (d‐2‐hydroxyacid dehydrogenase) protein family. Its crystal structure was determined by phase combination to 1.98 Å resolution. Structure–function relationships obtained by the comparison of HGDH with other members of the d‐2‐hydroxyacid dehydrogenase family give a chemically satisfying view of the substrate stereoselectivity and catalytic requirements for the hydride transfer reaction. A model for substrate recognition and turnover is discussed. The HGDH active site architecture is structurally optimized to recognize and bind the negatively charged substrate 2‐oxoglutarate. The structural position of the side chain of Arg52, and its counterparts in other family members, strongly correlates with substrate specificity towards substitutions at the C3 atom (linear or branched substrates). Arg235 interacts with the substrate's α‐carboxylate and carbonyl groups, having a dual role in both substrate binding and activation, and the γ‐carboxylate group can dock at an arginine cluster. The proton‐relay system built up by Glu264 and His297 permits His297 to act as acid–base catalyst and the 4Re‐hydrogen from NADH is transferred as hydride to the carbonyl group Si‐face leading to the formation of the correct enantiomer (R)‐2‐hydroxyglutarate.

Dehydratases catalyze the breakage of a carbon—oxygen bond leading to unsaturated products via the elimination of water. The 1.6-Å resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the γ-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of dehydratases with an unprecedented active site architecture. A [4Fe–4S]²⁺ cluster, coordinated by three cysteine and one histidine residues, is located 7 Å from the Re-side of a flavin adenine dinucleotide (FAD) moiety. The structure provides insight into the function of these ubiquitous prosthetic groups in the chemically nonfacile, radical-mediated dehydration of 4-hydroxybutyryl-CoA. The substrate can be bound between the [4Fe–4S]²⁺ cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversion. Our results raise interesting questions regarding the mechanism of acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, and address the divergent evolution of the ancestral common gene.

Uroporphyrinogen-III decarboxylase from Nicotiana tabacum is a plastidial enzyme involved in the biosynthesis of chlorophyll and haem. Sedimentation equilibrium with protein producing diffracting crystals clearly indicates that the enzyme is a homodimer under similar ionic strength conditions to those found in the chloroplast stroma. Additionally, dynamic light scattering reveals an ionic strength dependence for this oligomerization state. Crystals were obtained in the hexagonal space group P622 with one molecule per asymmetric unit and diffracted to 2.3 A resolution using synchrotron radiation.