In green species, sucrose can help antagonize abiotic stress. Sucrose phosphate synthase (SPS) is a well-known rate-limiting enzyme in the synthesis of sucrose. To date, however, there is no known crystal structure of SPS from plant or cyanobacteria. In this study, we report the first co-crystal structure of SPS from Thermosynechococcus elongatus with UDP and sucrose-6-phosphate (S6P). Within the catalytic site, the side chains of His158 and Glu331, along with two phosphate groups from UDP, form hydrogen bonds with the four hydroxyl groups of the glucose moiety in S6P. This association causes these four hydroxyl groups to become partially negatively charged, thus promoting formation of the C1 oxocarbenium ion. Breakage of the hydrogen bond between His158 and one of the hydroxyl groups may trigger covalent bond formation between the C1 oxocarbenium ion and the C2 hydroxyl of fructose-6-phosphate. Consistent with our structural model, we observed that two SPS mutants, H158A and E331A, lost all catalytic activity. Moreover, electron density of residues from two loops (loop1 and loop2) in the SPS A-domain was not observed, suggest their dynamic nature. B-factor analysis and molecular dynamics stimulations of the full-length enzyme and A-domain indicate that both loops are crucial for binding and release of substrate and product. In addition, temperature gradient analysis shows that SPS exhibits its highest activity at 70 • C, suggesting that this enzyme has the potential of being used in industrial production of S6P.
All living things have pyrophosphatases that hydrolyze pyrophosphate and release energy. This energetically favorable reaction drives many energetically unfavorable reactions. An accepted catalytic model of pyrophosphatase shows that a water molecule activated by two divalent cations (M1 and M2) within the catalytic center can attack pyrophosphate in an SN2 mechanism and thus hydrolyze the molecule. However, our co-crystal structure of Acinetobacter baumannii pyrophosphatase with pyrophosphate shows that a water molecule from the solvent may, in fact, be the actual catalytic water. In the co-crystal structure of the wild-type pyrophosphatase with pyrophosphate, the electron density of the catalytic centers of each monomer are different from one another. This indicates that pyrophosphates in the catalytic center are dynamic. Our mass spectroscopy results have identified a highly conserved lysine residue (Lys30) in the catalytic center that is phosphorylated, indicating that the enzyme could form a phosphoryl enzyme intermediate during hydrolysis. Mutation of Lys30 to Arg abolished the activity of the enzyme. In the structure of the apo wild type enzyme, we observed that a Na+ ion is coordinated by residues within a loop proximal to the catalytic center. Therefore, we mutated three key residues within the loop (K143R, P147G, and K149R) and determined Na+ and K+-induced inhibition on their activities. Compared to the wild type enzyme, P147G is most sensitive to these cations, whereas K143R was inactive and K149R showed no change in activity. These data indicate that monovalent cations could play a role in down-regulating pyrophosphatase activity in vivo. Overall, our results reveal new aspects of pyrophosphatase catalysis and could assist in the design of specific inhibitors of Acinetobacter baumannii growth.
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