Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C.

Abstract

Mycobacterium tuberculosis virulence is highly metal-dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu2+ ≥ Zn2+ > Ni2+ > Co2+ ) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for kcat rather than KM . Removal of the artificial N-terminal 6xHis-tag did not change the metal-dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8Å and 2.0Å resolution in the presence and absence of Zn2+ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal-dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.