Catalysis and inhibition of Mycobacterium tuberculosis methionine aminopeptidase

Abstract

Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.

Figure 1

Activation of MtMetAP1c apoenzyme by divalent metals.

Figure 2

Complementation of EcMetAP function by MtMetAP1c. A. E. coli cells carrying an amber mutation in chromosomal EcMetAP gene were streaked on agar plates with glucose (bottom plate) or with arabinose (to plate). Each plate displays cells containing pFLAGCTC (top), pFLAGCTC-MtMetAP1c (bottom left) or pFLAGCTC-EcMetAP (bottom right). B. Growth of the E. coli cells in liquid medium supplemented with glucose.

Figure 3

Structure of MtMetAP1c in the Mn(II)-form in complex with the Mn(II)-form selective inhibitor 4. A. Trimeric arrangement in crystal. Inhibitor 4 at the active site is shown as sticks. The three molecules of MtMetAP1c were colored cyan, magenta and yellow, respectively. B. Overlay of this structure with EcMetAP in complex with the same inhibitor (carbon magenta, pdb 1XNZ) and with the same protein in complex with methionine (carbon cyan, pdb 1YJ3). Only residues (thin sticks) surrounding the ligands (thick sticks) at the active site are shown. Non-carbon atoms are colored oxygen red, nitrogen blue, sulfur yellow, and chlorine green. Mn(II) (green) and Co(II) (red) ions are shown as spheres. For residue labeling, the first is for MtMetAP1c and the second for EcMetAP.

Figure 4

Structures of MtMetAP1c in the Ni(II)-form in complex with the Co(II)- and Ni(II)-form selective inhibitors 7 and 8. A. Trimetalated active site with 7 bound. Inhibitor is shown as thick sticks and the protein residues as thin sticks (carbon cyan, oxygen red, nitrogen blue, sulfur yellow, and fluorine pale). Ni(II) ions (yellow) are shown as large spheres, and water (red) and chlorine ion (green) are shown as small spheres. Metal coordination is shown as dashed lines. B. Dimetalated active site with 8 bound. Same color scheme, except carbon magenta and chlorine green. C. Comparison of the bound conformations of 7 and 8. For clarity, only selected protein residues are shown.