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Sialyltransferases are a family of glycosyltransferases that catalyze the transfer of N-acetylneuraminic acid residues from cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc) as a donor substrate to the carbohydrate groups of glycoproteins and glycolipids as acceptor substrates. We determined the crystal structure of Delta16psp26ST, the N-terminal truncated form of alpha2,6-sialyltransferase from Vibrionaceae Photobacterium sp. JT-ISH-224, complexed with a donor product CMP and an acceptor substrate lactose. Delta16psp26ST has three structural domains. Domain 1 belongs to the immunoglobulin-like beta-sandwich fold, and domains 2 and 3 form the glycosyltransferase-B structure. The CMP and lactose were bound in the deep cleft between domains 2 and 3. In the structure, only Asp232 was within hydrogen-binding distance of the acceptor O6 carbon of the galactose residue in lactose, and His405 was within hydrogen-binding distance of the phosphate oxygen of CMP. Mutation of these residues greatly decreased the activity of the enzyme. These structural and mutational results indicated that Asp232 might act as a catalytic base for deprotonation of the acceptor substrate, and His405 might act as a catalytic acid for protonation of the donor substrate. These findings are consistent with an in-line-displacement reaction mechanism in which Delta16psp26ST catalyzes the inverting transfer reaction. Unlike the case with multifunctional sialyltransferase (Delta24PmST1) complexed with CMP and lactose, the crystal structure of which was recently reported, the alpha2,6 reaction specificity of Delta16psp26ST is likely to be determined by His123.

Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a beta-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.

Many lines of evidence reveal that bisphenol A (BPA) functions at very low doses as an endocrine disruptor. The human estrogen-related receptor gamma (ERR gamma) behaves as a constitutive activator of transcription, although the endogenous ligand is unknown. We have recently demonstrated that BPA binds strongly to ERR gamma (K(D) = 5.5 nM), but not to the estrogen receptor (ER). BPA preserves the ERR gamma's basal constitutive activity, and protects the selective ER modulator 4-hydroxytamoxifen from its deactivation of ERR gamma. In order to shed light on a molecular mechanism, we carried out the X-ray analysis of crystal structure of the ERR gamma ligand-binding domain (LBD) complexed with BPA. BPA binds to the receptor cavity without changing any internal structures of the pocket of the ERR gamma-LBD apo form. The hydrogen bonds of two phenol-hydroxyl groups, one with both Glu275 and Arg316, the other with Asn346, anchor BPA in the pocket, and surrounding hydrophobic bonds, especially with Tyr326, complete BPA's strong binding. Maintaining the 'activation helix' (helix 12) in an active conformation would as a result preserve receptor constitutive activity. Our results present the first evidence that the nuclear receptor forms complexes with the endocrine disruptor, providing detailed molecular insight into the interaction features.

Ribonuclease NT (RNase NT), induced upon tobacco mosaic virus (TMV) infection in Nicotiana glutinosa leaves, has a broad base specificity. The crystal structures of RNase NT in complex with either 5'-AMP, 5'-GMP, or 2'-UMP were determined at 1.8 A resolutions by molecular replacement. RNase NT consists of seven helices and seven beta strands, and the structure is highly similar to that of RNase NW, a guanylic acid preferential RNase from the N. glutinosa leaves, showing root mean square deviation (rmsd) of 1.1 A over an entire length of two molecules for Calpha atoms. The complex structures revealed that Trp42, Asn44, and Trp50 are involved in interactions with bases at B1 site (primary site), whereas Gln12, Tyr17, Ser78, Leu79, and Phe89 participate in recognition of bases at B2 site (subsite). The 5'-GMP and 5'-AMP bind both B1 and B2 sites in RNase NT, while 2'-UMP predominantly binds B1 site in the complex. The nucleotide binding modes in these complexes would provide a clue to elucidation of structural basis for the broad base specificity for RNase NT.

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the removal of 5' leader sequences from tRNA precursors (pre-tRNA). The human protein Rpp21 is essential for human RNase P activity in tRNA processing in vitro. The crystal structure of Ph1601p from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, the archaeal homologue of Rpp21, was determined using the multiple anomalous dispersion (MAD) method with the aid of anomalous scattering in zinc and selenium at 1.6 A resolution. Ph1601p comprises an N-terminal domain (residues 1-55), a central linker domain (residues 56-79), and a C-terminal domain (residues 80-120), forming an L-shaped structure. The N-terminal domain consists of two long alpha-helices, while the central and C-terminal domains fold in a zinc ribbon domain. The electrostatic potential representation indicates the presence of positively charged clusters along the L arms, suggesting a possible role in RNA binding. A single zinc ion binds the well-ordered binding site that consists of four Cys residues (Cys68, Cys71, Cys97, and Cys100) and appears to stabilize the relative positions of the N- and C-domains. Mutations of Cys68 and Cys71 or Cys97 and Cys100 to Ser destabilize the protein structure, which results in inactivation of the RNase P activity. In addition, site-directed mutagenesis suggests that Lys69 at the central loop and Arg86 and Arg105 at the zinc ribbon domain are strongly involved in the functional activity, while Arg22, Tyr44, Arg65, and Arg84 play a modest role in the activity.

The crystal structure of alginate (poly alpha-l-guluronate) lyase from Corynebacterium sp. (ALY-1) was determined at 1.2A resolution using the MAD method and bromide ions. The structure of ALY-1 is abundant in beta-strands and has a deep cleft, similar to the jellyroll beta-sandwich found in 1,3-1,4-beta-glucanase. The structure suggests that alginate molecules may penetrate into the cleft to interact with the catalytic site of ALY-1. The reported crystal structure of another type of alginate lyase, A1-III, differs from that of ALY-1 in that it consists almost entirely of alpha-helical structure. Nevertheless, the putative catalytic residues in both enzymes are positioned in space in nearly identical arrangements. This finding suggests that both alginate lyases may have evolved through convergent evolution.

Ribonuclease NW (RNase NW), the wound-inducible RNase in Nicotiana glutinosa leaves, preferentially cleaves guanylic acid. We expressed the cDNA encoding RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant RNase NW (ryRNaseNW) secreted into medium was purified to apparent homogeneity using column chromatography. The crystal structure of ryRNase NW bound to 5'-GMP was determined at 1.5 A resolution by molecular replacement with tomato RNase LE as a search model. The RNase NW structurally belongs to the (alpha + beta) class of proteins, having eight helices (five alpha-helices and three 3(10) helices) and six beta-strands, and its structure is highly similar to those of other plant RNases, including a uridylic acid preferential RNase MC1 from bitter gourd seeds. The guanine ring of 5'-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: the guanine base is sandwiched between aromatic side chains of Tyr17 and Phe89. In addition, the guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79. Therefore, Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of the guanine base by RNase NW, findings which provide insight into the manner in which RNase NW preferentially cleaves guanylic acid.

Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to biliverdin by utilizing O(2) and NADPH. HO (apoHO) was crystallized as twinned P3(2) with three molecules per asymmetric unit, and its crystal structure was determined at 2.55 A resolution. Structural comparison of apoHO and its complex with heme (HO-heme) showed three distinct differences. First, the A helix of the eight alpha-helices (A-H) in HO-heme, which includes the proximal ligand of heme (His25), is invisible in apoHO. In addition, the B helix, a portion of which builds the heme pocket, is shifted toward the heme pocket in apoHO. Second, Gln38 is shifted toward the position where the alpha-meso carbon of heme is located in HO-heme. Nepsilon of Gln38 is hydrogen-bonded to the carbonyl group of Glu29 located at the C-terminal side of the A helix in HO-heme, indicative that this hydrogen bond restrains the angle between the A and B helices in HO-heme. Third, the amide group of Gly143 in the F helix is directed outward from the heme pocket in apoHO, whereas it is directed toward the distal ligand of heme in HO-heme. This means that the F helix around Gly143 must change its conformation to accommodate heme binding. The apoHO structure has the characteristic that the helix on one side of the heme pocket fluctuates, whereas the rest of the structure is similar to that of HO-heme, as observed in such hemoproteins as myoglobin and cytochromes b(5) and b(562). These structural features of apoHO suggest that the orientation of the proximal helix and the position of His25 are fixed upon heme binding.

RecJ, a 5' to 3' exonuclease specific for single-stranded DNA, functions in DNA repair and recombination systems. We determined the crystal structure of RecJ bound to Mn(2+) ion essential for its activity. RecJ has a novel fold in which two domains are interconnected by a long helix, forming a central groove. Mn(2+) is located on the wall of the groove and is coordinated by conserved residues characteristic of a family of phosphoesterases that includes RecJ proteins. The groove is composed of residues conserved among RecJ proteins and is positively charged. These findings and the narrow width of the groove indicate that the groove binds single- instead of double-stranded DNA.