SNAPSHOT OF A PHOSPHORYLATED SUBSTRATE INTERMEDIATE BY KINETIC CRYSTALLOGRAPHY

The ATP-dependent enzyme dethiobiotin synthetase from Escherichia coli catalyses the formation of dethiobiotin from CO2 and 7,8-diaminopelar gonic acid. The reaction is initiated by the formation of a carbamate and proceeds through a phosphorylated intermediate, a mixed carbamic p hosphoric anhydride, Here, we report the crystal structures at 1,9- an d 1.6-Angstrom resolution, respectively, of the enzyme-MgATP-diaminope largonic acid and enzyme-MgADP-carbamic-phosphoric acid anhydride comp lexes, observed by using kinetic crystallography, Reaction initiation by addition of either NaHCO3 or diaminopelargonic acid to crystals alr eady containing cosubstrates resulted in the accumulation of the phosp horylated intermediate at the active site. The phosphoryl transfer ste p shows inversion of the configuration at the phosphorus atom, consist ent with an in-line attack by the carbamate oxygen onto the phosphorus atom of ATP. A key feature in the structure of the complex of the enz yme with the reaction intermediate is two magnesium ions, bridging the phosphates at the cleavage site. These magnesium ions compensate the negative charges at both phosphate groups after phosphoryl transfer an d contribute to the stabilization of the reaction intermediate.