The enzyme urate oxidase catalyzes the conversion of uric acid to 5-hydroxyisourate, one of the steps in the ureide pathway. Arthrobacter globiformis urate oxidase (AgUOX) was crystallized and structures of crystals soaked in the substrate uric acid, the inhibitor 8-azaxanthin and allantoin have been determined at 1.9-2.2 A resolution. The biological unit is a homotetramer and two homotetramers comprise the asymmetric crystallographic unit. Each subunit contains two T-fold domains of betabetaalphaalphabetabeta topology, which are usually found in purine- and pterin-binding enzymes. The uric acid substrate is bound tightly to the enzyme by interactions with Arg180, Leu222 and Gln223 from one subunit and with Thr67 and Asp68 of the neighbouring subunit in the tetramer. In the other crystal structures, lithium borate, 8-azaxanthin and allantoate are bound to the enzyme in a similar manner as uric acid. Based on these AgUOX structures, the enzymatic reaction mechanism of UOX has been proposed.

Eukaryotic poly(A)-binding protein (PABP) binds not only to the 3' poly(A) tail of most mRNAs, but also to the 5'-untranslated region (UTR). The latter form of binding leads to auto-regulation of PABP expression. The 5'-UTR sequence contains A-rich repeats different from that in 3'-UTR. The role of these A-rich repeats in auto-regulation, however, remains unknown. To confirm that the 5'-UTR sequence has a specific structure before or after PABP binding, and to determine the function of such structure, several RNA/DNA fragments containing A-rich repeats were examined by crystallization, fluorescence microscopy and X-ray diffraction. Crystals were obtained for several of these fragments. In particular, single crystals were obtained for the DNA fragment with four repeats, suggesting that such fragment is folded into a regular structure through A:A interactions.

The crystal structures of pyruvate oxidase from Aerococcus viridans (AvPOX) complexed with flavin adenine dinucleotide (FAD), with FAD and thiamine diphosphate (ThDP) and with FAD and the 2-acetyl-ThDP intermediate (AcThDP) have been determined at 1.6, 1.8 and 1.9 A resolution, respectively. Each subunit of the homotetrameric AvPOX enzyme consists of three domains, as observed in other ThDP-dependent enzymes. FAD is bound within one subunit in the elongated conformation and with the flavin moiety being planar in the oxidized form, while ThDP is bound in a conserved V-conformation at the subunit-subunit interface. The structures reveal flexible regions in the active-site tunnel which may undergo conformational changes to allow the entrance of the substrates and the exit of the reaction products. Of particular interest is the role of Lys478, the side chain of which may be bent or extended depending on the stage of catalysis. The structures also provide insight into the routes for electron transfer to FAD and the involvement of active-site residues in the catalysis of pyruvate to its products.

Oligonucleotides containing 5-(N-aminohexyl)carbamoyl-modified uracils have promising features for applications as antigene and antisense therapies. Relative to unmodified DNA, oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (NU) or 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (NUm), respectively exhibit increased binding affinity for DNA and RNA, and enhanced nuclease resistance. To understand the structural implications of NU and NUm substitutions, we have determined the X-ray crystal structures of DNA:DNA duplexes containing either NU or NUm and of DNA:RNA hybrid duplexes containing NUm. The aminohexyl chains are fixed in the major groove through hydrogen bonds between the carbamoyl amino groups and the uracil O4 atoms. The terminal ammonium cations on these chains could interact with the phosphate oxygen anions of the residues in the target strands. These interactions partly account for the increased target binding affinity and nuclease resistance. In contrast to NU, NUm decreases DNA binding affinity. This could be explained by the drastic changes in sugar puckering and in the minor groove widths and hydration structures seen in the NUm containing DNA:DNA duplex structure. The conformation of NUm, however, is compatible with the preferred conformation in DNA:RNA hybrid duplexes. Furthermore, the ability of NUm to render the duplexes with altered minor grooves may increase nuclease resistance and elicit RNase H activity.

D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between D-3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate D-3-hydroxybutyrate and the cofactor NAD(+) at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD(+) and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of D-3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure.