File:Substrate Catalytic Triad.jpg

2015-04-24T19:22:06Z

Ben Zercher:

Sandbox Reserved 1053

2015-04-24T19:19:54Z

Ben Zercher:

{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}
== Background ==

The antigen 85 (ag85) complex in [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''], which is responsible for causing the disease [http://en.wikipedia.org/wiki/Tuberculosis Tuberculosis], is composed of three intracellular membrane proteins: Ag85A, B, and C. The Ag85 complex is a major component of the cell wall, with each protein catalyzing the transfer of important cell wall constituents into the membrane. <ref>PMID: 10655617</ref> The cell wall of ''Mycobacterium tuberculosis'' is composed of three primary molecules: [http://en.wikipedia.org/wiki/Peptidoglycan peptidoglycans], [http://en.wikipedia.org/wiki/Arabinogalactan arbinogalactans], and [http://en.wikipedia.org/wiki/Mycolic_acid mycolic acids]. Ag85C is of particular interest due to its transfer of mycolic acids, which is one of the major components in determining cell wall integrity. The mycolic acids are responsible for forming the outermost layer of the cell wall. Mycolic acids have a long fatty acid chain and exhibit extreme hydrophobicity, which effectively creates a hydrophobic envelope surrounding the bacterium. The hydrophobic envelope created by they mycolic acids creates a barrier against small hydrophilic molecules, such as Tuberculosis antibiotics. By targeting this mycoloyltransferase activity, inhibition of Ag85C offers potential for cell wall disruption and subsequent antibiotic targeting for normally drug-resistant ''Mycotaberia tuberculosis''. <ref>PMID: 10200974</ref>
==Structure==
<StructureSection load='1dqz' size='400' side='right' caption='Antigen 85C in ''Mycobacterium Tuberculosis''' scene=''>

== General Structure ==

[[Image:Substrate Binding 2D Surface.jpg|200 px|left|thumb|Octylthioglucoside, a substrate analog, shown in the binding pocket of Antigen 85C]]

Antigen 85C was crystallized in its dimeric form.<ref>PMID: 25028518</ref> The <scene name='69/694220/Secondary_structures/2'>secondary structure</scene> shown in the monomeric form is composed of helices, shown in pink, with one interwoven beta sheet, shown in yellow. The confrontation of a central β-sheet bordered by α–helices creates an a α/β hydrolase fold in Ag85C, and this tertiary conformation is highly conserved across enzymes that function in this capacity. <ref>PMID:10655617</ref> The substrate binding pocket of Ag85C is composed of two separate but equally important components; there is carbohydrate binding pocket for the trehalose, and there is a fatty acid binding pocket for the mycolic acid. As a result, trehalose monomycolate can effectively bind to the Ag85C binding pocket. This binding pocket is shown in the left image with a substrate mimic.

== Enzymatic Activity ==

Mutagenesis studies have confirmed the Ag85C functions through a Glu-His-Ser <scene name='69/694220/Catalytic_triad/4'>catalytic triad</scene>, similar to that of [http://en.wikipedia.org/wiki/Chymotrypsin 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 (Figure #). The S124 alcohol’s nucleophilicity is inductively strengthened through H260 and E224, which allows the S124 residue to catalyze a reaction that involves [http://en.wikipedia.org/wiki/Cord_factor 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 the figure below.

[[Image:Mech_Ag85C.jpeg|500 px|center|General reaction catalyzed by Antigen 85C]]

== Methods of Inhibition ==

[[Image:C209.jpeg|200 px|left|thumb|Cys209 stabilizing kinked formation of alpha-9 helix]]

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 C209 and L232-T 231 (Figure #). The C209 facilitated interaction causes the <scene name='69/694220/Alpha_9_helix/2'>α9 helix</scene> 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.

[[Image:Ebselen_inhibition.jpeg|200 px|right|thumb|Ebselen inhibition relaxing the alpha-9 helix]]

Ag85C can be inhibited by [http://en.wikipedia.org/wiki/Ebselen ebselen] covalently bound to the sulfur of C209. Ebselen is a thiol-modifying agent that serves as an electrophile for the C209 that results in a sulfur-selenium bond. 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-T31, which effectively disrupts the interaction that holds the α9 helix in the active conformation. Furthermore, the bulk of ebselen creates steric hindrance with the α9 helix residues (Figure #). Relaxation of the α9 helix removes E228 and H260, which now interacts with S148, from the active site. The absence of these residues decreases the nucleophilicity of the S124 alcohol which decreases serine hydrolytic activity.

Relaxed alpha helix scene <scene name='69/694220/Inhibited_relaxed_helix/1'>relaxed</scene>

===Inhibitors===

Additional thiol-modifying agents, [http://en.wikipedia.org/wiki/4-Chloromercuribenzoic_acid p-chloromercuribenzoic acid] and [http://en.wikipedia.org/wiki/Iodoacetamide 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.

[[Image:Inhibitors_Ag85c.jpeg|400 px|center]]

</StructureSection>

== References ==
<references/>

2015-04-17T17:52:49Z

Ben Zercher:

{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}
== Background ==

The antigen 85 (ag85) complex in [[''Mycobacterium tuberculosis'']] is composed of three intracellular membrane proteins: Ag85A, B, and C. The Ag85 complex is a major component of the cell wall, with each protein catalyzing the transfer of important cell wall constituents into the membrane. <ref>PMID: 10655617</ref> Ag85C is of particular interest due to its transfer of mycolic acids, which are major components in determining cell wall integrity. By targeting this mycoloyltransferase activity, inhibition of Ag85C offers potential for cell wall disruption and subsequent antibiotic targeting for normally drug-resistant ''Mycotaberia tuberculosis''. <ref>PMID: 10200974</ref>
==Structure==
<StructureSection load='1dqz' size='400' side='right' caption='Antigen 85C in ''Mycobacterium Tuberculosis''' scene=''>

== General Structure ==

Antigen 85C was crystallized in its dimeric form.<ref>PMID: 25028518</ref> The <scene name='69/694220/Secondary_structures/2'>secondary structure</scene> shown in the monomeric form is composed of helices, shown in pink, with one interwoven beta sheet, shown in yellow. The confrontation of a central β-sheet bordered by α–helices creates an a α/β hydrolase fold in Ag85C, and this tertiary conformation is highly conserved across enzymes that function in this capacity. <ref>PMID:10655617</ref> The substrate binding pocket of Ag85C is composed of two separate but equally important components; there is carbohydrate binding pocket for the trehalose, and there is a fatty acid binding pocket for the mycolic acid. As a result, trahalose monomycolate can effectively bind to the Ag85C binding pocket.

== Enzymatic Activity ==

Mutagenesis studies have confirmed the Ag85C functions through a Glu-His-Ser <scene name='69/694220/Catalytic_triad/4'>catalytic triad</scene>, 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 (Figure #). 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.

[[Image:General_mechanism.jpg|400 px|center|General reaction catalyzed by Antigen 85C]]

== Methods of Inhibition ==

[[Image:C209.jpeg|200 px|left|thumb|Cys209 stabilizing kinked formation of alpha-9 helix]]

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 C209 and L232-T 231 (Figure #). The C209 facilitated interaction causes the <scene name='69/694220/Alpha_9_helix/2'>α9 helix</scene> 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.>

[[Image:Ebselen_inhibition.jpeg|200 px|right|thumb|Ebselen inhibition relaxing the alpha-9 helix]]

Ag85C can be inhibited by ebselen covalently bound to the sulfur of C209. Ebselen is a thiol-modifying agent that serves as an electrophile for the C209 that results in a sulfur-selenium bond. 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-T31, which effectively disrupts the interaction that holds the α9 helix in the active conformation. Furthermore, the bulk of ebselen creates steric hindrance with the α9 helix residues (Figure #). Relaxation of the α9 helix removes E228 and H260, which now interacts with S148, from the active site. The absence of these residues decreases the nucleophilicity of the S124 alcohol which decreases serine hydrolytic activity.

Relaxed alpha helix scene <scene name='69/694220/Inhibited_relaxed_helix/1'>relaxed</scene>

===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.

[[Image:Inhibitors_Ag85c.jpeg|400 px|center]]

</StructureSection>

== References ==
<references/>