Sandbox Reserved 1074

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This Sandbox is Reserved from 02/09/2015, through 05/31/2016 for use in the course "CH462: Biochemistry 2" taught by Geoffrey C. Hoops at the Butler University. This reservation includes Sandbox Reserved 1051 through Sandbox Reserved 1080.

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Enoyl-ACP Reductase InhA from Mycobacterium tuberculosis

1 Introduction
- 1.1 FAS-II System
- 1.2 Mechanism of Action
2 General Structural Information
- 2.1 Ligands
3 Fatty Acyl Binding Crevice
- 3.1 Substrate Binding Loop Flexibility
- 3.2 Importance of Tyr-158
4 Catalytic Triad
5 Clinical Applications

Introduction

FAS-II System

Mechanism of Action

General Structural Information

Crystal structures of InhA reveal a (each subunit featured with a different color) in aqueous solution with separate ligand binding sites in each subunit ^[1]. Each subunit is composed of 289 residues and features a typical Rossmann fold containing a single NADH binding site. The of InhA is made up of several alpha helices (pink), beta sheets (gold), and beta turns (white) ^[2]. This enzyme also features a fatty acyl binding crevice that accommodates the long-chain fatty acyl substrate needed to synthesize mycolic acid precursors.

Fatty Acyl Binding Crevice (substrate binding loop in purple; substrates pictured inside the crevice)

The of the InhA form one side of the fatty acyl binding crevice, referred to as the (residues 196-219). One side of this crevice is open and exposed to solvent, which allows the substrates to access the binding pocket of this enzyme. The size of the substrate binding loop is a primary determinant of the ability of InhA to select for fatty acyl chains longer than 16 carbons to successfully produce mycolic acid precursors ^[1].

Ligands

The two ligands that play a role in the function of InhA are NADH and the long-chain fatty acyl substrate. NADH, or nicotinamide adenine dinucleotide, is a cofactor found in all living cells. It is especially important in the function of InhA because it helps position the fatty acyl substrate within the fatty acyl binding crevice, thereby improving the overall activity of InhA. Once properly positioned, the fatty acyl substrates are reduced at the trans double bond between C2 and C3, forming mycolic acid precursors that eventually compose the cell wall of mycobacteria ^[1].

Fatty Acyl Binding Crevice

Within the fatty acyl binding crevice, the NADH substrate sits on the top shelf of the Rossmann fold, and the fatty acyl substrate sits on top of the NADH substrate. Due to its position within the crevice, the trans double bond of the fatty acyl substrate is found near the closed end of the crevice and is located directly adjacent to the nicotinamide ring of NADH.

Substrate Binding Pocket (NADH in green; fatty acyl substrate in red)

These two molecules interact with each other via hydrogen bonding. The first hydrogen bond occurs between the phosphate oxygen of NADH and the amide nitrogen of the N-acetylcysteamine portion of the fatty acyl substrate. The second hydrogen bond occurs between the 2'-hydroxyl of the nicotinamide ribose of NADH and the thioester carbonyl oxygen of the fatty acyl substrate ^[1].

Substrate Binding Loop Flexibility

In addition to the hydrogen bonding that occurs between the NADH and fatty acyl substrate within the crevice, each of these molecules is also held in place within the crevice through interactions with the side chains of surrounding (purple) residues. The majority of these residues anchoring the substrates are found within the substrate binding loop itself, including Ala-198, Met-199, Ala-201, Ile-202, Leu-207, Ile-215, and Leu-218. Additional hydrophobic amino acids that are not a part of the substrate binding loop also play a role in positioning and stabilizing the fatty acyl chain in the crevice. Studies have found that the fatty acyl substrate adopts a u-shaped conformation to facilitate binding. As the fatty acyl substrate binds in the crevice, the substrate binding loop shifts outward toward the solvent, and the Tyr-158 residue is rotated to facilitate the binding of fatty acyl chains of 16 carbons or greater. During this conformational change upon substrate binding, no hydrogen bonds are broken, which supports the flexibility of the substrate binding loop. It is likely that this flexibility of the substrate binding loop provides increased freedom for the active site to accept fatty acyl substrates of varying carbon chain lengths ^[1].

Importance of Tyr-158

One example of a hydrophobic amino acid that is not a part of the substrate binding loop yet interacts with the fatty acyl substrate is Tyr-158.

Tyr-158 (light blue) hydrogen bonding to the fatty acyl substrate, 2TK (red)

This amino acid is conserved in other enoyl-ACP reductases in both bacteria and plants, so it likely plays an essential role in the function of these specific enzymes. Studies have shown that Tyr-158 forms the only direct hydrogen bond that exists between the InhA protein and the fatty acyl substrate. This hydrogen bond occurs between the hydroxyl oxygen on the side chain of Tyr-158 and the thioester carbonyl oxygen of the fatty acyl substrate. Consequently, the Tyr-158 residue acts to stabilize the enolate intermediate that forms during the hydride transfer reaction ^[1].

Catalytic Triad

Structural studies have shown that InhA possesses a , composed of Phe-149, Tyr-158, and Lys-165. This catalytic triad of InhA is analogous to the classic Ser-Tyr-Lys catalytic triad present in all members of the SDR (Short-chain Dehydrogenase Reductase) family ^[1]. In the dehydrogenases, the first catalytic residue is usually Ser or Thr, while in the enoyl reuctases (InhA), the first catalytic residue is usually Phe or Tyr ^[2]. According to crystallographic data, the likely role of Tyr-158 is to stabilize the enolate intermediate that forms during the hydride transfer reaction ^[1]. Previous structural and kinetic studies have confirmed that the side chain of the Lys-165 residue in InhA functions similarly to the catalytic Lys residues in other SDR enzymes. The catalytic Lys-165 residue in InhA interacts with the 2' or 3'-hydroxyls of the nicotinamide ring of NADH to hold this cofactor in place within the fatty acyl binding crevice ^[1]. Finally, the exact role of the catalytic Phe-149 residue in InhA is less well known. Recent Raman spectroscopy studies have supported that the catalytic Phe-149 residue plays an essential role in orienting the NADH cofactor correctly to lower the energy of the transition state and promoting the hydride transfer reaction ^[2]. Altogether, the residues in the catalytic triad of InhA play critical roles in properly orienting NADH and the fatty acyl substrate to promote the hydride transfer reaction required to elongate the fatty acyl chains and produce mycolic acid precursors.

Clinical Applications

References

↑ ^1.0 ^1.1 ^1.2 ^1.3 ^1.4 ^1.5 ^1.6 ^1.7 ^1.8 Rozwarski, D.A. (1999). Crystal Structure of the Mycobacterium tuberculosis Enoyl-ACP Reductase, InhA, in Complex with NAD⁺ and a C16 Fatty Acyl Substrate. Journal of Biological Chemistry, 274(22), 15582-15589. PMID: 10336454 DOI: 10.1074/jbc.274.22.15582
↑ ^2.0 ^2.1 ^2.2 Bell, A.F. (2007). Evidence from Raman Spectroscopy That InhA , the Mycobacterial Enoyl Reductase, Modulates the Conformation of the NADH Cofactor to Promote Catalysis. Journal of the American Chemical Society, 129, 6425-6431. DOI: 10.1021/ja068219m

Retrieved from "http://52.214.119.220/wiki/index.php/Sandbox_Reserved_1074"

@@ Line 18: / Line 18: @@
 ==='''Ligands'''===
-The two ligands that play a role in the function of InhA are [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NADH] and the long-chain fatty acyl substrate. NADH, or nicotinamide adenine dinucleotide, is a cofactor found in all living cells. It is especially important in the function of InhA because it helps position the fatty acyl substrate within the fatty acyl binding crevice, thereby improving the overall activity of InhA. Once properly positioned, the fatty acyl substrates are reduced at the ''trans'' double bond between C2 and C3, forming mycolic acid precursors that eventually compose the cell wall of mycobacteria.
+The two ligands that play a role in the function of InhA are [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NADH] and the long-chain fatty acyl substrate. NADH, or nicotinamide adenine dinucleotide, is a cofactor found in all living cells. It is especially important in the function of InhA because it helps position the fatty acyl substrate within the fatty acyl binding crevice, thereby improving the overall activity of InhA. Once properly positioned, the fatty acyl substrates are reduced at the ''trans'' double bond between C2 and C3, forming mycolic acid precursors that eventually compose the cell wall of mycobacteria <ref name="InhA" />.
 == '''Fatty Acyl Binding Crevice''' ==
-Within the fatty acyl binding crevice, the NADH substrate sits on the top shelf of the Rossmann fold, and the fatty acyl substrate sits on top of the NADH substrate. Due to its position within the crevice, the ''trans'' double bond of the fatty acyl substrate is found near the closed end of the crevice and is located directly adjacent to the nicotinamide ring of [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NADH].  [[Image:Binding Pocket - Mesh.jpg|thumb|250px|right|Substrate Binding Pocket (NADH in green; fatty acyl substrate in red)]] These two molecules interact with each other via hydrogen bonding. The first hydrogen bond occurs between the phosphate oxygen of NADH and the amide nitrogen of the N-acetylcysteamine portion of the fatty acyl substrate. The second hydrogen bond occurs between the 2'-hydroxyl of the nicotinamide ribose of NADH and the thioester carbonyl oxygen of the fatty acyl substrate.
+Within the fatty acyl binding crevice, the NADH substrate sits on the top shelf of the Rossmann fold, and the fatty acyl substrate sits on top of the NADH substrate. Due to its position within the crevice, the ''trans'' double bond of the fatty acyl substrate is found near the closed end of the crevice and is located directly adjacent to the nicotinamide ring of [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NADH].  [[Image:Binding Pocket - Mesh.jpg|thumb|250px|right|Substrate Binding Pocket (NADH in green; fatty acyl substrate in red)]] These two molecules interact with each other via hydrogen bonding. The first hydrogen bond occurs between the phosphate oxygen of NADH and the amide nitrogen of the N-acetylcysteamine portion of the fatty acyl substrate. The second hydrogen bond occurs between the 2'-hydroxyl of the nicotinamide ribose of NADH and the thioester carbonyl oxygen of the fatty acyl substrate <ref name="InhA" />.
 ==='''Substrate Binding Loop Flexibility''' ===
-In addition to the hydrogen bonding that occurs between the NADH and fatty acyl substrate within the crevice, each of these molecules is also held in place within the crevice through interactions with the side chains of surrounding <scene name='69/694241/Sbl_hydrophobic/1'>hydrophobic</scene> (purple) residues. The majority of these <scene name='69/694241/Hydrophobic_residues/1'>hydrophobic</scene> residues anchoring the substrates are found within the substrate binding loop itself, including Ala-198, Met-199, Ala-201, Ile-202, Leu-207, Ile-215, and Leu-218. Additional hydrophobic amino acids that are not a part of the substrate binding loop also play a role in positioning and stabilizing the fatty acyl chain in the crevice. Studies have found that the fatty acyl substrate adopts a u-shaped conformation to facilitate binding. As the fatty acyl substrate binds in the crevice, the substrate binding loop shifts outward toward the solvent, and the [[Tyr-158]] residue is rotated to facilitate the binding of fatty acyl chains of 16 carbons or greater. During this conformational change upon substrate binding, no hydrogen bonds are broken, which supports the flexibility of the substrate binding loop. It is likely that this flexibility of the substrate binding loop provides increased freedom for the active site to accept fatty acyl substrates of varying carbon chain lengths.
+In addition to the hydrogen bonding that occurs between the NADH and fatty acyl substrate within the crevice, each of these molecules is also held in place within the crevice through interactions with the side chains of surrounding <scene name='69/694241/Sbl_hydrophobic/1'>hydrophobic</scene> (purple) residues. The majority of these <scene name='69/694241/Hydrophobic_residues/1'>hydrophobic</scene> residues anchoring the substrates are found within the substrate binding loop itself, including Ala-198, Met-199, Ala-201, Ile-202, Leu-207, Ile-215, and Leu-218. Additional hydrophobic amino acids that are not a part of the substrate binding loop also play a role in positioning and stabilizing the fatty acyl chain in the crevice. Studies have found that the fatty acyl substrate adopts a u-shaped conformation to facilitate binding. As the fatty acyl substrate binds in the crevice, the substrate binding loop shifts outward toward the solvent, and the [[Tyr-158]] residue is rotated to facilitate the binding of fatty acyl chains of 16 carbons or greater. During this conformational change upon substrate binding, no hydrogen bonds are broken, which supports the flexibility of the substrate binding loop. It is likely that this flexibility of the substrate binding loop provides increased freedom for the active site to accept fatty acyl substrates of varying carbon chain lengths <ref name="InhA" />.
 ==='''Importance of Tyr-158'''===
-One example of a hydrophobic amino acid that is not a part of the substrate binding loop yet interacts with the fatty acyl substrate is [[Tyr-158]]. [[Image:Tyr-158.jpg|thumb|200px|left|Tyr-158 (light blue) hydrogen bonding to the fatty acyl substrate, 2TK (red)]] This amino acid is conserved in other enoyl-ACP reductases in both bacteria and plants, so it likely plays an essential role in the function of these specific enzymes. Studies have shown that Tyr-158 forms the only direct hydrogen bond that exists between the InhA protein and the fatty acyl substrate. This hydrogen bond occurs between the hydroxyl oxygen on the side chain of Tyr-158 and the thioester carbonyl oxygen of the fatty acyl substrate. Consequently, the Tyr-158 residue acts to stabilize the enolate intermediate that forms during the hydride transfer reaction (1).
+One example of a hydrophobic amino acid that is not a part of the substrate binding loop yet interacts with the fatty acyl substrate is [[Tyr-158]]. [[Image:Tyr-158.jpg|thumb|200px|left|Tyr-158 (light blue) hydrogen bonding to the fatty acyl substrate, 2TK (red)]] This amino acid is conserved in other enoyl-ACP reductases in both bacteria and plants, so it likely plays an essential role in the function of these specific enzymes. Studies have shown that Tyr-158 forms the only direct hydrogen bond that exists between the InhA protein and the fatty acyl substrate. This hydrogen bond occurs between the hydroxyl oxygen on the side chain of Tyr-158 and the thioester carbonyl oxygen of the fatty acyl substrate. Consequently, the Tyr-158 residue acts to stabilize the enolate intermediate that forms during the hydride transfer reaction <ref name="InhA" />.
 == '''Catalytic Triad''' ==
-Structural studies have shown that InhA possesses a <scene name='69/694241/Catalytic_triad/2'>Phe-Tyr-Lys catalytic triad</scene>, composed of Phe-149, Tyr-158, and Lys-165. This catalytic triad of InhA is analogous to the classic Ser-Tyr-Lys catalytic triad present in all members of the SDR (Short-chain Dehydrogenase Reductase) family (1). In the dehydrogenases, the first catalytic residue is usually Ser or Thr, while in the enoyl reuctases (InhA), the first catalytic residue is usually Phe or Tyr (2). According to crystallographic data, the likely role of Tyr-158 is to stabilize the enolate intermediate that forms during the hydride transfer reaction (1). Previous structural and kinetic studies have confirmed that the side chain of the Lys-165 residue in InhA functions similarly to the catalytic Lys residues in other SDR enzymes. The catalytic Lys-165 residue in InhA interacts with the 2' or 3'-hydroxyls of the nicotinamide ring of NADH to hold this cofactor in place within the fatty acyl binding crevice (1). Finally, the exact role of the catalytic Phe-149 residue in InhA is less well known. Recent Raman spectroscopy studies have supported that the catalytic Phe-149 residue plays an essential role in orienting the NADH cofactor correctly to lower the energy of the transition state and promoting the hydride transfer reaction (2). Altogether, the residues in the catalytic triad of InhA play critical roles in properly orienting NADH and the fatty acyl substrate to promote the hydride transfer reaction required to elongate the fatty acyl chains and produce mycolic acid precursors.
+Structural studies have shown that InhA possesses a <scene name='69/694241/Catalytic_triad/2'>Phe-Tyr-Lys catalytic triad</scene>, composed of Phe-149, Tyr-158, and Lys-165. This catalytic triad of InhA is analogous to the classic Ser-Tyr-Lys catalytic triad present in all members of the SDR (Short-chain Dehydrogenase Reductase) family <ref name="InhA" />. In the dehydrogenases, the first catalytic residue is usually Ser or Thr, while in the enoyl reuctases (InhA), the first catalytic residue is usually Phe or Tyr <ref name="Phe149" />. According to crystallographic data, the likely role of Tyr-158 is to stabilize the enolate intermediate that forms during the hydride transfer reaction <ref name="InhA" />. Previous structural and kinetic studies have confirmed that the side chain of the Lys-165 residue in InhA functions similarly to the catalytic Lys residues in other SDR enzymes. The catalytic Lys-165 residue in InhA interacts with the 2' or 3'-hydroxyls of the nicotinamide ring of NADH to hold this cofactor in place within the fatty acyl binding crevice <ref name="InhA" />. Finally, the exact role of the catalytic Phe-149 residue in InhA is less well known. Recent Raman spectroscopy studies have supported that the catalytic Phe-149 residue plays an essential role in orienting the NADH cofactor correctly to lower the energy of the transition state and promoting the hydride transfer reaction <ref name="Phe149" />. Altogether, the residues in the catalytic triad of InhA play critical roles in properly orienting NADH and the fatty acyl substrate to promote the hydride transfer reaction required to elongate the fatty acyl chains and produce mycolic acid precursors.