Polyphosphate Kinase Terminal Modifications Alter Enzymatic Activity and Affect Stress Recovery in E. coli

Many bacterial species, including Escherichia coli (E. coli), utilize the enzyme polyphosphate kinase (PPK) to synthesize polyphosphate (polyP) in response to biological stresses. Multiple studies have shown that impairing PPK activity impairs bacterial pathogenicity and survival. Further, no PPK homolog has been identified in mammalian species, making it a promising therapeutic target. While the structure, kinetics, and mechanism of PPK is well‐characterized, its regulation is poorly understood. We previously described a series of mutations in E. coli ppk , termed ppk* , that result in high polyP accumulation in vivo . However, the specific activity of PPK* enzyme in vitro , when purified using a C‐term 6X His‐tag (PPK*‐HT), was comparable to purified WT enzyme (PPK‐HT). Previous reports have indicated that N‐term modifications impair PPK activity. We hypothesized that the C‐term His tag alters the in vitro activity. To test this, we compared the specific activities of PPK purified using a smaller C‐term C tag (PPK‐CT) and the C‐term His tag enzymes to a natively purified PPK enzyme with no tags and a detagged PPK purified using the N‐term Profinity tag (PPK*‐DT and PPK‐DT). We found that the PPK*‐HT and PPK‐HT maintained comparable activities, but were significantly more active than the other purified enzymes. PPK‐CT and native PPK had comparable activities, suggesting the smaller C‐tag does not alter PPK activity. Both PPK*‐DT and PPK‐DT had drastically reduced activity, likely due to two N‐term residues left behind by the Profinity elution process. PPK*‐DT had very low activity, while the PPK‐DT enzyme had no detectable activity, suggesting that the PPK* mutation does increase the specific activity in the absence of a C‐term His tag. As polyP synthesis is heavily involved in bacterial stress responses that reduce growth and replication under unfavorable conditions, we hypothesized that a more active PPK would impair recovery following a prolonged stationary phase. To determine if the C‐term modifications affect bacterial growth and recovery, we developed strains carrying one of four expression vectors: an empty vector control (EV), PPK‐HT, PPK‐CT, or native PPK. Using these strains, we measured the effect of tagged PPK on recovery from stationary phase over 24 hours. We found that while the PPK‐CT and native PPK strains had comparable recovery and and shifted into log phase at roughly the same time, the PPK‐CT strain had a modestly slower recovery resulting in a later shift into log phase. Importantly, the empty vector control had the fastest recovery rate, which is consistent with our understanding of polyP synthesis acting to impair growth. Taken together, our results have significant implications for the field of polyP biology. We have shown that the current field‐wide purification system produces a more active PPK, which results in both in vivo and in vitro differences compared to native PPK. In the future we will focus on understanding how these modifications affect PPK's activity, whether the termini represent novel PPK regulatory elements, and whether the C‐tag can be used to purify native‐comparable PPK in place of the widely‐used His‐tag.