The 440-amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168-residue mini-intein, ΔI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (ΔI-CM) increased C-terminal cleavage activity. Using the I-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139-residue mini-intein, ΔΔI hh -SM, by replacing 36 amino acids in the residual endonuclease loop with a seven-residue β-turn from the autoprocessing domain of Hedgehog protein. The threedimensional structures of ΔI-SM, ΔΔI hh -SM, and two variants, ΔΔI hh -CM and ΔΔI hh , have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N-and C-termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side chain of Asp422 plays a role in both N-and C-terminal cleavage, thereby suggesting that this highly-conserved residue regulates the balance between the two reactions.

The tetramer observed in the crystal is likely to be physiologically relevant. Because the Mg(2+)-oxalate is inaccessible to solvent, substrate binding and dissociation might be accompanied by conformational changes. A mechanism involving a phosphoenzyme intermediate is proposed, with Asp58 acting as the nucleophilic entity that accepts and delivers the phosphoryl group. The active-site architecture and the chemistry performed by PEP mutase are different from other alpha/beta-barrel proteins that bind pyruvate or PEP, thus the enzyme might represent a new family of alpha/beta-barrel proteins.

The flavivirus methyltransferase (MTase) sequentially methylates the N7 and 2’-O positions of the viral RNA cap (GpppA-RNA→m7GpppA-RNA→m7GpppAm-RNA), using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here the synthesis and biological evaluation of a series of novel nucleoside analogs. Two of these compounds can effectively and competitively inhibit the WNV MTase with IC50 values in micromolar range and, more importantly, do not inhibit human MTase. The compounds can also suppress the WNV replication in cell culture.