Substrate orientation in 4-oxalocrotonate tautomerase and its effect on QM/MM energy profiles

The tautomerization of 2-oxo-4E-hexendioate by 4-oxalocrotonate tautomerase has been studied by quantum mechanical/molecular mechanical (QM/MM) methods using three models, A−C, with different substrate orientations. The computed QM/MM energy profiles are rather different. Various energy partitioning analyses indicate the origin of these differences and the role of the active site residues for different substrate orientations. The proposed new model C is preferred over the previously used models A and B because it combines favorable substrate binding geometries with reasonable barriers and is consistent with the experimental evidence from mutation studies concerning the catalytic ability of specific residues in the binding site, especially R11‘

Identification and characterization of a selenoprotein family containing a diselenide bond in a redox motif

Selenocysteine (Sec, U) insertion into proteins is directed by translational recoding of specific UGA codons located upstream of a stem-loop structure known as Sec insertion sequence (SECIS) element. Selenoproteins with known functions are oxidoreductases containing a single redox-active Sec in their active sites. In this work, we identified a family of selenoproteins, designated SelL, containing two Sec separated by two other residues to form a UxxU motif. SelL proteins show an unusual occurrence, being present in diverse aquatic organisms, including fish, invertebrates, and marine bacteria. Both eukaryotic and bacterial SelL genes use single SECIS elements for insertion of two Sec. In eukaryotes, the SECIS is located in the 3′ UTR, whereas the bacterial SelL SECIS is within a coding region and positioned at a distance that supports the insertion of either of the two Sec or both of these residues. SelL proteins possess a thioredoxin-like fold wherein the UxxU motif corresponds to the catalytic CxxC motif in thioredoxins, suggesting a redox function of SelL proteins. Distantly related SelL-like proteins were also identified in a variety of organisms that had either one or both Sec replaced with Cys. Danio rerio SelL, transiently expressed in mammalian cells, incorporated two Sec and localized to the cytosol. In these cells, it occurred in an oxidized form and was not reducible by DTT. In a bacterial expression system, we directly demonstrated the formation of a diselenide bond between the two Sec, establishing it as the first diselenide bond found in a natural protein