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Running title: Structures of mammalian glutamine synthetases Data deposition: The atomic coordinates and structure factors have been deposited at the Protein Data Bank, www.pdb.org (PDB ID codes 2UU7, 2OJW and 2QC8). * Manuscript 2 SummaryGlutamine synthetase catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in glutamine synthetase activity and neurodegenerative disorders such as Alzheimer's. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. Glutamine synthetase is also a central component of bacterial nitrogen metabolism, and a potential drug target.Previous studies had investigated the structures of bacterial and plant glutamine synthetases. In the present publication, we report the first structures of mammalian glutamine synthetases. The apo form of the canine enzyme was solved by molecular replacement, and refined to a resolution of 3 Å. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 Å and 2.6 Å, respectively.Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial as well as eukaryotic glutamine synthetases, while the nucleotide-binding site varies to a much larger degree. Thus the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting glutamine synthetase must be designed with caution.

Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic kinetic resolution of racemic glycidyl phenyl ether. Kinetic data, molecular dynamics calculations, molecular modeling, inhibition experiments and X-ray structural work for the wild-type (WT) enzyme and the best mutant reveal the basis of the large increase in enantioselectivity (E = 4.6 versus E = 115). The overall structures of the WT and the mutant are essentially identical, but dramatic differences are observed in the active site as revealed by the X-ray structures. All of the experimental and computational results support a model in which productive positioning of the preferred (S)-glycidyl phenyl ether, but not the (R)-enantiomer, forms the basis of enhanced enantioselectivity. Predictions regarding substrate scope and enantioselectivity of the best mutant are shown to be possible.