D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD Bp ) at a 2.7-Å resolution. The structure of HYD Bp consists of a core (␣/␤) 8 triose phosphate isomerase barrel fold and a ␤-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYD Bp shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYD Tsp ) and Bacillus stearothermophilus (HYD Bst ), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs. The more thermostable HYD Tsp contains more aromatic residues in the interior of the structure than HYD Bp and HYD Bst . Changes of large aromatic residues in HYD Tsp to smaller residues in HYD Bp or HYD Bst decrease the hydrophobicity and create cavities inside the structure. HYD Tsp has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD Bp and HYD Bst . Besides, HYD Tsp also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.Dihydropyrimidinases (EC 3.5.2.2) are a family of enzymes that catalyze the reversible hydrolytic ring opening of the amide bond in five-or six-membered cyclic diamides (54, 62). In humans, they are responsible for the hydrolysis of dihydropyrimidines at the second step of reductive catabolism of pyrimidines. The homologous enzymes in microorganisms are hydantoinases (HYDs) which catalyze the hydrolysis of 5-substituted hydantoins to the enantiomerically pure Ncarbamyl amino acids. The latter can be converted chemically or enzymatically to the corresponding optically pure amino acids (50, 54, 64). In the biotechnology industry, HYD is widely used in the production of D-amino acids, including D-p-hydroxyphenylglycine, which are the precursors for semisynthesis of antibiotics, peptide hormones, pyrethroids, and pesticides (3,49,53,54). Depending on their stereoselectivities and specificities on substrates, HYDs are often classified as D-, L-, or non-enantio specific (47, 67).One major consideration in the application of enzymes for biocatalysis is thermostability. Screening of various thermophiles has successfully discovered several thermostable D-HYDs from Thermus sp. CBS30380 and Lu1220 (28), and Bacillus stearothermophilus SD-1 (38) and GH-2 (51). Substantial efforts...

The liver receptor homolog 1 (LRH-1) belongs to the Fushi tarazu factor 1 nuclear receptor subfamily, and its biological functions are just being unveiled. The molecular mechanism for the transcriptional regulation by LRH-1 is not clear yet. In this report, we use mutagenesis and reporter gene assays to carry out a detailed analysis on the hinge region and the proximal ligand binding domain (LBD) of human (h) LRH-1 that possess important regulatory functions. Our results indicate that helix 1 of the LBD is essential for the activity of hLRH-1 and that the steroid receptor coactivator (SRC)-1 interacts directly with the LBD of hLRH-1 and significantly potentiates the transcriptional activity of hLRH-1. Cotransfection assays demonstrate that overexpressed SRC-1 potentiates hLRH-1 mediated activation of the cholesterol 7-alpha-hydroxylase promoter and increases the transcription of the endogenous cholesterol 7-alpha-hydroxylase in Huh7 cells. The interaction between SRC-1 and hLRH-1 assumes a unique pattern that involves primarily a region containing the glutamine-rich domain of SRC-1, and helix 1 and activation function-2 of hLRH-1 LBD. Mutagenesis and molecular modeling studies indicate that, similar to mouse LRH-1, the coactivator-binding cleft of hLRH-1 LBD is not optimized. An interaction between helix 1 of hLRH-1 LBD and a region containing the glutamine-rich domain of SRC-1 can provide an additional stabilizing force and enhances the recruitment of SRC-1. Similar interaction is observed between hLRH-1 and SRC-2/transcriptional intermediary factor 2 or SRC-3/acetyltransferase. Moreover, transcriptional intermediary factor 2 and acetyltransferase also potentiate the transcriptional activity of hLRH-1, suggesting a functional redundancy among SRC family members. These findings collectively demonstrate an important functional role of helix 1 in cofactor recruitment and reveal a novel molecular mechanism of transcriptional regulation and cofactor recruitment mediated by hLRH-1.

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNA(Leu). The editing domain of LeuRS deacylates the mischarged Ile-tRNA(Leu) and Met-tRNA(Leu). We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 A, 2.4 A, and 3.2 A resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.