Ca(II) is not a MetAP activator and does not inhibit purified MetAP enzyme. coli BL21(DE3) cells and used the recombinant MetAP still inside live bacterial cells as the enzyme reagent. Therefore, we conclude that Fe(II) is the likely metal used by MetAP in E. We confirmed their cellular target as MetAP by analysis of N-terminal processed and unprocessed recombinant glutathione S-transferase proteins. Further, we found that only these Fe(II)-form selective inhibitors showed significant inhibition of growth of five E. We observed that only inhibitors that are selective for the Fe(II)-form of MetAP were potent in this assay. 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. Therefore, the challenge is to elucidate the physiologically relevant metal for MetAP and discover MetAP inhibitors that can effectively inhibit cellular MetAP. 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. Many MetAP inhibitors are highly potent on purified enzyme, but they fail to show significant inhibition of bacterial growth. Although purified enzyme can be activated by several divalent metal ions, the exact metal ion used by MetAP in cells is unknown. Being an essential enzyme for bacteria, MetAP is an appealing target for the development of novel antibacterial drugs. Glycobiology and Extracellular Matricesĭivalent metal ions play a critical role in the removal of N-terminal methionine from nascent proteins by methionine aminopeptidase (MetAP).Mohamadi, F., Richards, N.G.J., Guida, W.C., Liskamp, R., Lipton, M., Caufield, C., Chang, G., Hendrickson, T. Zhan, C.-G., de Souza, O.N., Rittenhouse, R. Lowther, W.T., Zhang, Y., Sampson, P.B., Honek, J.F. Tahirov, T.H., Oki, H., Tsukihara, T., Ogasahara, K., Yutani, K., Ogata, K., Izu, Y., Tsunasawa, S. Liu, S., Widom, J., Kemp, C.W., Crews, C.M. Lowther, W.T., Orville, A.M., Madden, D.T., Lim, S., Rich, D.H. Griffith, E.C., Su, Z., Niwayama, S., Ramsay, C.A., Chang, Y.-H. Lowther, W.T., McMillen, D.A., Orville, A.M. Griffith, E.C., Su, Z., Turk, B.E., Chen, S., Chang, Y.-H., Wu, Z., Biemann, K. By contrast, the nature of the oxygen bridging the metal ions within the metal binding site of eMetAP-1 cannot be determined based on the results here, due to the similar structural results obtained with a bridging water molecule and a bridging hydroxide ion.įolkman, J., N. Within the site of hMetAP-2 the results strongly indicate that a hydroxide ion bridges the metal ions. Another important structural issue is the identity of the bridging oxygen, which is part of either a water molecule or a hydroxide ion. colimethionine aminopeptidase type 1 (eMetAP-1). This was the case for both of the systems studied one based on the X-ray structure of the human methionine aminopeptidase type 2 (hMetAP-2) and the other based on the X-ray structure of the E. The calculations showed that the structure of the site was not influenced by the identity of the metal ions. The structure of this site with either Co 2+ions or Zn 2+ions was investigated using density functional theory. All methionine aminopeptidases exhibit the same conserved metal binding site.