Folate derivatives are essential vitamins for cell growth and replication, primarily because of their central role in reactions of one-carbon metabolism. Folates require polyglutamation to be efficiently retained within the cell and folate-dependent enzymes have a higher affinity for the polyglutamylated forms of this cofactor. Polyglutamylation is dependent on the enzyme folylpolyglutamate synthetase (FPGS), which catalyzes the sequential addition of several glutamates to folate. FPGS is essential for the growth and survival of important bacterial species, including Mycobacterium tuberculosis, and is a potential drug target. Here, the crystal structures of M. tuberculosis FPGS in complex with ADP and AMPPCP are reported at 2.0 and 2.3 angstroms resolution, respectively. The structures reveal a deeply buried nucleotide-binding site, as in the Escherichia coli and Lactobacillus casei FPGS structures, and a long extended groove for the binding of folate substrates. Differences from the E. coli and L. casei FPGS structures are seen in the binding of a key divalent cation, the carbamylation state of an essential lysine side chain and the adoption of an 'open' position by the active-site beta5-alpha6 loop. These changes point to coordinated events that are associated with dihydropteroate/folate binding and the catalysis of the new amide bond with an incoming glutamate residue.

Archaerhodopsin-2 (aR2), the sole protein found in the claret membrane of Halorubrum sp. Aus-2, functions as a light-driven proton pump. In this study, structural analysis of aR2 was performed using a novel three-dimensional crystal prepared by the successive fusion of claret membranes. The crystal is made up of stacked membranes, in each of which aR2 trimers are arranged on a hexagonal lattice. This lattice structure resembles that found in the purple membrane of H. salinarum, except that lipid molecules trapped within the trimeric structure are not distributed with perfect threefold symmetry. Nonetheless, diffraction data at 1.8 Å resolution provide accurate structural information about functionally important residues. It is shown that two glutamates in the proton-release channel form a paired structure that is maintained by a low-barrier hydrogen bond. Although the structure of the proton-release pathway is highly conserved among proton-pumping archaeal rhodopsins, aR2 possesses the following peculiar structural features: (i) the motional freedom of the tryptophan residue that makes contact with the C13 methyl group of retinal is restricted, affecting the formation/decay kinetics of the L state, and (ii) the N-terminal polypeptide folds into an Ω-loop, which may play a role in organizing the higher-order structure.