DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD ؉ and assistance of a divalent metal cofactor Mg 2؉ . Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover 31 P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged Mg 2؉ , and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4 -5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations.chemical movie ͉ nucleotidyl transfer ͉ structural reaction kinetics ͉ time-resolved cryo-magic-angle-spinning NMR ͉ transition state U nderstanding chemical mechanics of biocatalysis is a fundamental goal of life sciences. With the development of high-resolution x-ray diffraction analysis and solution NMR a large number of protein structures in the resting state have been solved, giving knowledge on how proteins look, e.g., the detailed view of protein architecture on the primary, secondary, tertiary, and quaternary structure levels. Further insight is coming with studies on how proteins work, e.g., by observing changes of the protein structure in the course of a chemical reaction-recording a molecular movie. Femtosecond laser pulses, molecular beams and ultrafast electron diffraction are used in (in)organic chemistry for monitoring breaking and forming of chemical bonds in real time (1). In biochemistry, kinetic crystallography is used to record molecular movies, an approach that combines starting and stopping the reaction in a protein crystal with x-ray data acquisition at low temperatures (2-8). The successful outcome of a time-resolved x-ray experiment depends as much on a prompt triggering of the reaction as on preparing well diffracting protein crystals. Here we introduce time-resolved lowtemperature magic-angle-spinning (cryo-MAS) NMR spectroscopy as a complementary ''noninvasive'' technique to study catalytic dynamics of biochemical reactions. It combines phototriggering and freeze-trapping with real-time monitoring of chemical transformations and requires neither protein crystallization nor high intensity penetrating radiation beams. We have assayed the nucleotidyl transfer reaction catalyzed by DNA ligase from bacteriophage T4, a Mg 2ϩ -and AT...

Nitrogen fixation (NF) potential was measured in more than 40 samples of soda solonchak soils with the pH of water extract between 9.5 and 11.0 collected in several locations of Central Asia and in Egypt, using the acetylene reduction method. NF was detected in most of the samples. Maximal rates were observed under microaerophilic-anaerobic conditions with glucose as a substrate. In most cases, the NF negatively correlated with salt content and alkalinity. Five enrichments at pH 10 under micro-oxic conditions with glucose resulted in stable haloalkaliphilic mixed cultures, with diazotrophic component(s) active up to 2.0-3.0 M total Na(+). The cultures were dominated by Gram-positive spore-forming bacteria. Molecular cloning of nifH genes demonstrated the presence of two phylogenetic lineages of diazotrophs in the enrichments affiliated with the low-GC Gram-positive bacteria (in rRNA groups 1 and 6 of bacilli and in Clostridiales). Isolation of pure cultures of haloalkaliphilic diazotrophs from micro-oxic enrichments yielded nine strains, comprising two phylogenetic lineages. Most of the isolates (eight) were affiliated with the aerotolerant fermentative haloalkaliphilic bacterium Amphibacillus tropicus and a single strain clustered with the obligately anaerobic haloalkaliphile Bacillus arseniciselenatis. Diazotrophy has never been recognized previously in these groups of Gram-positive bacteria. Overall, the results demonstrated the existence, in soda solonchak soils, of a novel group of free-living fermentative diazotrophic bacteria active at extremely haloalkaline conditions.

The light-driven intramolecular redox reaction of adenosine-5'-triphosphate-[P3-(1-(2-nitrophenyl)-ethyl)]ester (caged ATP) has been studied in frozen aqueous solution using time-resolved solid state NMR spectroscopy under continuous illumination conditions. Cleavage of the phosphate ester bond leads to 0.3, 1.36, and 6.06 ppm downfield shifts of the alpha-, beta-, and gamma-phosphorus resonances of caged ATP, respectively. The observed rate of ATP formation is 2.4 +/- 0.2 h(-1) at 245 K. The proton released in the reaction binds to the triphosphate moiety of the nascent ATP, causing the upfield shifts of the 31P resonances. Analyses of the reaction kinetics indicate that bond cleavage and proton release are two sequential processes in the solid state, suggesting that the 1-hydroxy,1-(2-nitrosophenyl)-ethyl carbocation intermediate is involved in the reaction. The beta-phosphate oxygen atom of ATP is protonated first, indicating its proximity to the reaction center, possibly within hydrogen bonding distance. The residual linewidth kinetics are interpreted in terms of chemical exchange processes, hydrogen bonding of the beta-phosphate oxygen atom and evolution of the hydrolytic equilibrium at the triphosphate moiety of the nascent ATP. Photoreaction of caged ATP in situ gives an opportunity to study structural kinetics and catalysis of ATP-dependent enzymes by NMR spectroscopy in rotating solids.

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