The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxidoreductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at

Beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 A resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 A). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an alpha-helix and a beta-strand I with 17.91 A in length, while the span of vitamin D(3) is about 12.51 A. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).

Substrate inhibition is a characteristic feature of many cytosolic sulfotransferases. The differences between the complex structures of SULT2A1/DHEA and SULT2A1/PAP or SULT2A1/ADT (Protein Data Bank codes are 1J99, 1EFH, and 1OV4, respectively) have enabled us to elucidate the specific amino acids responsible for substrate inhibition. Based on the structural analyses, substitution of the smaller residue alanine for Tyr-238 (Y238A) significantly increases the K i value for dehydroepiandrosterone (DHEA) and totally eliminates substrate inhibition for androsterone (ADT). In addition, Met-137 was proposed to regulate the binding orientations of DHEA and ADT in SULT2A1. Complete elimination or regeneration of substrate inhibition for SULT2A1 with DHEA or ADT as substrate, respectively, was demonstrated with the mutations of Met-137 on Y238A mutant. Analysis of the Met-137 mutants and Met-137/ Tyr-238 double mutants uncovered the relationship between substrate binding orientations and inhibition in SULT2A1. Our data indicate that, in the substrate inhibition mode, Tyr-238 regulates the release of bound substrate, and Met-137 controls substrate binding orientation of DHEA and ADT in SULT2A1. The proposed substrate inhibition mechanism is further confirmed by the crystal structures of SULT2A1 mutants at Met-137. We propose that both substrate binding orientations exhibited substrate inhibition. In addition, a corresponding residue in other cytosolic sulfotransferases was shown to have a function similar to that of Tyr-238 in SULT2A1.

PDB Reference: Bacillus amyloliquefaciens -amylase, 3bh4.The crystal structure of Bacillus amyloliquefaciens -amylase (BAA) at 1.4 Å resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family. The final model of BAA is composed of two molecules in a back-to-back orientation, which is likely to be a consequence of crystal packing. Despite a high degree of identity, comparison of the structure of BAA with those of other liquefying-type -amylases indicated moderate discrepancies at the secondary-structural level. Moreover, a domain-displacement survey using anisotropic B-factor and domain-motion analyses implied a significant contribution of domain B to the total flexibility of BAA, while visual inspection of the structure superimposed with that of B. licheniformis -amylase (BLA) indicated higher flexibility of the latter in the central domain A. Therefore, it is suggested that domain B may play an important role in liquefying -amylases, as its rigidity offers a substantial improvement in thermostability in BLA compared with BAA.

-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D 3 binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D 3 to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was "disrupted" by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D 3 in the complex by altering the pH and initial ratios of vitamin D 3 /LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D 3 /LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D 3 in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.

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Cytosolic sulfotransferases (SULTs) are traditionally known as the Phase II drug-metabolizing or detoxifying enzymes that serve for the detoxification of drugs and other xenobiotics. These enzymes in general catalyze the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to low-molecular weight substrate compounds containing hydroxyl or amino group(s). Despite considerable efforts made in recent years, some fundamental aspects of the SULTs, particularly their ontogeny, cell type/tissue/organ-specific distribution, and physiological relevance, particularly their involvement in drug metabolism and detoxification, still remain poorly understood. To better understand these fundamental issues, we have embarked on developing the zebrafish as a model for studies concerning the SULTs. To date, fifteen zebrafish SULTs have been cloned, expressed, purified, and characterized. These zebrafish SULTs, which fall into four major SULT gene families, exhibited differential substrate specificities and distinct patterns of expression at different stages during embryogenesis, through larval development, and on to maturity. The information obtained, as summarized in this review, provides a foundation for further investigation into the physiological and pharmacological involvement of the SULTs using the zebrafish as a model.