Biological Role

Cell Wall of Mycobacteria

The unusually thick and waxy cell wall of mycobacteria is primarily composed of peptidoglycans, arbinogalactans, and mycolic acids. The mycolic acids, which have very long carbon chains and exhibit extreme hydrophobicity, are responsible for forming the outermost layer of the cell wall, thus creating a hydrophobic envelope surrounding the mycobacterium. Much of the mycolic acid content of the cell wall is in the form of esters (trehalose-6-monomycolate, TMM) and bis-esters (trehalose-6,6'-dimycolate, TDM, cord factor) of trehalose.

Enzymatic Activity

The antigen 85 (Ag85) complex in Mycobacterium tuberculosis is composed of three homologous proteins: Ag85A, Ag85B, and Ag85C, encoded by the fbpA, fbpB, and fbpC genes, respectively. Each of these Ag85 components (A, B, and C) is an acyltransferase enzyme (EC 2.3.1.122) that can catalyze mycolyl transfer from TMM to another alcoholic substrate, including TMM itself (Figure 1).

Figure 1: A mycolyl transfer reaction catalyzed by Ag85C

Clinical Relevance

The hydrophobic envelope created by these inclusion of mycolic acids in the cell walls of mycobacteria results in a barrier against small hydrophilic molecules, such as Tuberculosis antibiotics. Thus, inhibition of Ag85C offers potential for cell wall disruption and subsequent antibiotic targeting for normally drug-resistant Mycobacteria tuberculosis. ^[1]

Structure

Figure 2: Octylthioglucoside (green), a substrate analog, shown in the binding pocket of Ag85C (tan, from 1va5)

Wild type Ag85C, in the absence of any specific ligands, was originally crystallized as a .^[2] The (shown in the monomeric form) is 38% α-helical (pink) and 16% β-sheet (yellow). The confrontation of a central β-sheet bordered by α–helices comprises an α/β hydrolase fold in Ag85C, a supersecondary structure that is highly conserved across serine hydrolases. The active site of Ag85C is composed of two adjoined binding pockets; there is carbohydrate binding pocket for the trehalose substrate, and there is a fatty acid binding pocket for the mycolic acid. As a result, trehalose monomycolate can effectively bind to the Ag85C active site. This binding pocket is shown in Figure 2, in which Ag85C is shown with a bound substrate mimic, octylthioglucoside^[3] (Figure 3).

INSERT CHEMDRAW PICTURE COMPARING THE OCTYLTHIOGLYCOSIDE STRUCTURE TO TMM

Catalytic Triad

Figure 2: Relation of the catalytic triad to the octylthioglucoside analog in Ag85C

Mutagenesis studies^[4] have confirmed the Ag85C functions through a Glu-His-Ser , similar to that of chymotrypsin. By modifying each of the catalytic residues separately testing the enzyme’s relative activity, it has been shown that mutation of any one of these residues dramatically reduces activity. The S124 alcohol’s nucleophilicity is inductively strengthened through H260 and E224, which allows the S124 residue to catalyze a reaction that involves trehalose 6, 6’-dimycolate. The formation of the functional catalytic triad relies on upon Van der Waals interaction between C209 and the peptide bond between L232 and T231. This interaction results in a kinked conformation of the α9 helix, which promotes that activity of the catalytic triad. As a result, Ag85C, a mycolyl transferase, can facilitate the modification of trehalose monomycolates to trehalose dimycolates, which are then transported to the bacterial cell wall. This reaction is shown in Figure 3 below.

Figure 3: General reaction catalyzed by Antigen 85C

Methods of Inhibition

Figure 4: Cys209 stabilizing kinked formation of alpha-9 helix in the native Ag85C enzyme

Due to the importance of Ag85C enzymatic activity in maintaining the integrity of the Mycobacteria tuberculosis cell wall though mycolic acid modifications, the Ag85C enzyme represents a potentially effective avenue for inhibiting cell growth. The conformational sensitivity of the active site residues, H260, E228, and S124, relies entirely upon Van der Waals interaction between , which can be seen in both the scene and Figure 4. The C209 facilitated interaction causes the to acquire a kinked conformation that promotes optimal interaction distances between catalytic residues. As a result, C209 has been a specific target residue for Ag85C inhibition.

Figure 5: Ebselen inhibition relaxing the alpha-9 helix in Ag85C-Ebselen

Ag85C can be inhibited by ebselen, which covalently bounds to the sulfur in C209. Ebselen is a thiol-modifying agent that serves as an electrophile for a C209 nucleophilic attack that results in sulfur-selenium bond formation. Co-crystallization of ebselen with Ag85C provides an explanation for the mechanism of ebselen-based inhibition. The addition of ebselen increases the distance between C209 and L232-T231, which effectively disrupts the interaction that holds the α9 helix in the active conformation. The disruption of this interaction causes the α9 helix to . Furthermore, the bulk of ebselen creates steric hindrance with the α9 helix residues, which can be seen in Figure 5. The pink helix represents the native enzyme, and the tan helix represents Ag85C covalently bound to ebselen, which is shown in green. Relaxation of the α9 helix due to ebselen removes E228 and H260, which now interacts with S148, from the active site. The absence of these residues destroys the charge relay mechanism, and as a result, the nucleophilicity of the S124 alcohol is not longer strengthened, which decreases serine hydrolytic activity.

Inhibitors

Additional thiol-modifying agents, p-chloromercuribenzoic acid and iodoacetamide, were crystalized with Ag85C. The structures show that each of these thiol-reactive inhibitors covalently bound to C209 and caused a relaxation of the α9 helix in a similar fashion to ebselen.

Figure 6: Known thiol-reactive inhibitors of Ag85C