FE(II) is the native cofactor for Escherichia coli methionine aminopeptidase

Abstract

Divalent metal ions play a critical role in the removal of N-terminal methionine from nascent proteins by methionine aminopeptidase (MetAP). Being an essential enzyme for bacteria, MetAP is an appealing target for the development of novel antibacterial drugs. Although purified enzyme can be activated by several divalent metal ions, the exact metal ion used by MetAP in cells is unknown. Many MetAP inhibitors are highly potent on purified enzyme, but they fail to show significant inhibition of bacterial growth. One possibility for the failure is a disparity of the metal used in activation of purified MetAP and the metal actually used by MetAP inside bacterial cells. Therefore, the challenge is to elucidate the physiologically relevant metal for MetAP and discover MetAP inhibitors that can effectively inhibit cellular MetAP. We have recently discovered MetAP inhibitors with selectivity toward different metalloforms of Escherichia coli MetAP, and with these unique inhibitors, we characterized their inhibition of MetAP enzyme activity in a cellular environment. We observed that only inhibitors that are selective for the Fe(II)-form of MetAP were potent in this assay. Further, we found that only these Fe(II)-form selective inhibitors showed significant inhibition of growth of five E. coli strains and two Bacillus strains. We confirmed their cellular target as MetAP by analysis of N-terminal processed and unprocessed recombinant glutathione S-transferase proteins. Therefore, we conclude that Fe(II) is the likely metal used by MetAP in E. coli and other bacterial cells.

FIGURE 1.

Effect of Ca(II) on the cellular MetAP activity. E. coli cells were suspended in 50 mm Tris, and increasing amounts of CaCl₂ were added. MetAP enzyme activity was measured as hydrolysis of Met-AMC by fluorescence.

FIGURE 2.

Effect of inhibition of MetAP on N-terminal processing of recombinant GST protein. A, ESI-MS protonation multiplicity spectra of a mixture of processed and unprocessed GST with charge states ranging from +19 to +26. B, spectra were transformed to a mass scale. The mass difference between both peaks corresponds to a methionine residue. Positions for the processed and unprocessed GST proteins are indicated by arrows. In both A and B, the spectrum using the sample in the absence of 6 is shown on the top, and the spectrum using the sample in the presence of 6 is shown on the bottom.