Crystal Structures of Pyrophosphatase from Acinetobacter baumannii: Snapshots of Pyrophosphate Binding and Identification of a Phosphorylated Enzyme Intermediate

Abstract

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.

Covalent Adduct Formation in Methylthio-d-ribose-1-phosphate Isomerase: Reaction Intermediate or Artifact?

“…With no other candidate nucleophiles in the vicinity of the active site, it is increasingly likely that the observed phosphorylation is an artifact and/or occurs at a remote site with no obvious functional purpose. Artifactual phosphorylation of an aspartate residue by a phosphate ester has been reported for pyrophosphatase, , for example. It is interesting to note that oxocarbenium ion 3 (Scheme B,C) is expected to be an excellent phosphorylating agent considering the leaving group would be 4 , which is a relatively stable molecule.…”

Characterization of a family I inorganic pyrophosphatase from Legionella pneumophila Philadelphia 1

“…Other similar proteins included PPases from Acinetobacter baumannii (PDB entry 6k21, r.m.s.d. = 0.495 A ˚over 141 residues; Si et al, 2019) and Oleispira antarctica (PDB entry 3i4q, r.m.s.d. = 0.508 A ˚over 151 residues; Kube et al, 2013).…”

“…Major structural differences occur outside the OB-fold �-barrel and many of the catalytically significant residues are identical (Heikinheimo et al, 1996(Heikinheimo et al, , 2001Lahti et al, 1990;Kajander et al, 2013). In addition to high structural conservation between the bacterial monomers, the oligomeric architecture is also likely to be conserved as the PPases from E. coli and A. baumannii are confirmed to be hexamers (Harutyunyan et al, 1997;Si et al, 2019).…”