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) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k cat rather than K M . Removal of the artificial N-terminal 6xHis-tag did not change the metaldependent 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 1.8 Å and 2.0 Å resolution in the presence and absence of Zn 2+ 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 metaldependent 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.

Revisiting Jatropha curcas Monomeric Esterase: A Dienelactone Hydrolase Compatible with the Electrostatic Catapult Model

“…Similar effects were also observed for other plant esterases/lipases such as C. pepo [39], Glycine max, Oryza sativa [42], and those in wheat flour [43]. Metal ions can affect esterases/lipases and other α/β hydrolases activity by different mechanisms such as by coordinating with active site residues [44], overall structure alteration by allosteric regulation [44,45], surface potential alteration (a pH-dependent event) [46], and reaction equilibrium dislocation through low-soluble salt production with one of the hydrolysis reaction products, namely the carboxylic acid (mainly described for Ca 2+ [47]). Whether it is an activation or inhibition scenario depends on each enzyme-metal ion pair and this activity alteration usually represents a gain or loss of enzyme stability.…”

Sequence and Structural Motifs Controlling the Broad Substrate Specificity of the Mycobacterial Hormone-Sensitive Lipase LipN

“…The enzyme kinetic activity of LipN was assayed via the hydrolysis of the fluorogenic substrates (Figure ) in accordance with past kinetic analysis of mycobacterial serine hydrolases. ,,,, Kinetic assays were run in triplicate on black 96-well plates in a Synergy H1 Hybrid Reader (BioTek). Fluorogenic ester substrates (10 mM in DMSO) were diluted to a starting concentration of 100 μM in PBS containing acetylated BSA (Sigma; 0.1 mg/mL).…”

A conserved but structurally divergent loop in acyl protein thioesterase 1 regulates its catalytic activity, ligand binding, and folded stability

“…The enzyme kinetic activity of APT1 was assayed via the hydrolysis of the fluorogenic substrates (Figure 1) in accordance with past kinetic analysis of APT1 and FTT258. 23,[31][32][33][34] Kinetic assays were run in triplicate on 96-well plates in a Synergy H1 Hybrid Reader (BioTek).…”

“…Lid domains also provide potential dynamic components to the α/β hydrolase structure with transitions between open and closed states dependent on lipid binding. [20][21][22][23] APTs do not have a canonical lid or cap domain, but instead use a short loop to define a shallow, hydrophobic binding pocket. 13,18 In addition to human APTs, homologous APTs with overlapping functions are found in plants, protozoan parasites, and various bacterial species.…”

“…These lid and cap domains overhang or frame the substrate‐binding pocket and provide substrate selectivity for diverse intracellular substrates. Lid domains also provide potential dynamic components to the α/β hydrolase structure with transitions between open and closed states dependent on lipid binding 20–23 . APTs do not have a canonical lid or cap domain, but instead use a short loop to define a shallow, hydrophobic binding pocket 13,18 …”