Potassium channels allow K+ ions to easily diffuse through their pores while effectively preventing smaller Na+ ions from permeation. The ability to discriminate between these two similar and abundant ions is vital for these proteins to control electrical and chemical activity in all organisms. This selection process occurs at the narrow selectivity filter that contains structurally identified K+ binding-sites. Selectivity is thought to arise because smaller ions such as Na+ do not bind to these K+ sites in a thermodynamically favorable way. Using the model K+ channel KcsA, we examined how intracellular Na+ and Li+ interact with the pore and the permeant ions using electrophysiology, molecular dynamics simulations, and X-ray crystallography. Our results suggest that these small cations have a binding site within the K+ selectivity filter, albeit different from the K+ sites. We propose that selective permeation from the intracellular side is achieved mainly by a large energy barrier blocking filter entry for Na+ and Li+ in the presence of K+, and not by a difference of binding affinity between ions inside the selectivity filter.

A fluorescent probe has been attached to the carboxy terminus of the α-subunit of α,β-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of α-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophore's absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert ␣-D-ribose 5-phosphate (ribose 5-phosphate) and ␣-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Enzyme-catalyzed phosphoryl transfer forms the basis for many biological, bioenergetic, and regulatory processes and is one of the most common cellular reactions (1). Numerous enzyme families have evolved mechanistically distinct solutions for phosphoryl transfer (2). Phosphomutases are phosphotransfer enzymes that rearrange the position of phosphate within a substrate molecule through either intramolecular (i.e. the phosphate is transferred to a different position on the same molecule) or intermolecular phosphoryl transfer (i.e. the phosphate is transferred from one substrate molecule to another).Bacterial phosphopentomutases (PPMs) 3 (EC 5.4.2.7) interconvert ribose 1-phosphate and ribose 5-phosphate, which bridges glucose metabolism and RNA biosynthesis (3). The importance of this reaction has recently been underscored by the observation that targeted deletion of the gene encoding PPM in the pathogen Francisella tularensis (deoB) results in markedly decreased virulence (4). PPMs appear to be biochemically and structurally distinct from their human congeners (5, 6), making them potential targets for antibiotic development.Sequence clustering classifies prokaryotic PPMs within the alkaline phosphatase superfamily of metalloenzymes, which includes a range of functionally diverse enzymes such as cofactor-independent phosphoglycerate mutase, phosphodiesterase, and estrone and aryl sulfatases (7). The majority of alkaline phosphatase superfamily enzymes catalyze a hydrolase reaction; however, both PPM (5) and the cofactor-independent phosphoglycerate mutase catalyze phosphomutase reactions (8, 9).All previously characterized alkaline phosphatase superfamily members follow a unified general reaction mechanism ( Fig. 1) (10). In alkaline phosphatase itself (11, 12), the catalytically competent enzyme has an unphosphorylated catalytic nucleophile, Ser-102 (Fig. 1, state 1). Turnover is initiated when the metallocenter activates a phosphoester donor substrate (R D -OPO 3 H Ϫ ) (Fig. 1, state 2) to transfer the phosphoryl group to the hydroxyl of Ser-102 (Fig. 1, state 3). This results in a covalent phosphoenzyme intermediate (E-OPO 3 H Ϫ ) (Fig. 1, state 4). A second phosphoryl transfer from the enzyme to the acceptor water molecule (Fig. 1, states 5 and 6) completes the reaction cycle. This general reaction mechanism has also been verified * This work was supported, in whole or in part, by National Institutes of Health Grants GM079419 (to T. M. I.), GM077189 (to B. O. B.), GM051366 (to B. E. W.), DK070787 (to B. E. W.), T32 NS07491 (to T. D. P.), T32 GM008320 (to T. D. P.), T90 DA022873 (to D. P. N.), and T32 GM07628 (to G. R. W.). This work was also supported by a pilot award funded by the Vanderbilt Instit...

Concatenation of engineered biocatalysts into multistep pathways dramatically increases their utility, but development of generalizable assembly methods remains a significant challenge. Herein we evaluate ‘bioretrosynthesis’, which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2,3-dideoxyinosine). Applying both directed evolution and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and 3-fold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase, but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.