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{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE -->
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==Your Protein Name here==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
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=='''Enoyl-ACP Reductase InhA from ''Mycobacterium tuberculosis'''''==
== Biological Function ==
<StructureSection load='4OHU' size='340' side='right' caption='Enoyl-ACP Reductase InhA (PDB: [[4ohu]])' scene='69/694241/Homotetramer_publication/1'>


== Structural Overview ==


== '''Introduction''' ==
== Mechanism of Action ==


The Enoyl-ACP Reductase InhA, from [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''], catalyzes the [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NADH]-dependent reduction of long-chain ''trans''-2-enoyl-ACP fatty acids in the type II fatty acid biosynthesis pathway of ''M.'' ''tuberculosis''. InhA is a member of the [http://en.wikipedia.org/wiki/Short-chain_dehydrogenase short chain dehydrogenase/reductase] (SDR) family of enzymes. InhA is the only enoyl-ACP reductase found in tuberculosis, making the enzyme a potential drug target <ref name="Phe149" />.
== Zinc Ligand(s) ==


=== '''FAS-II System''' ===
== Other Ligands ==
 
[http://en.wikipedia.org/wiki/Mycolic_acid Mycolic acids] are very long-chain fatty acids (C<sub>60</sub> -C<sub>90</sub>) that are essential components of the mycobacterial cell wall. Mycolic acids are synthesized by at least two known elongation systems, type I and type II [http://en.wikipedia.org/wiki/Fatty_acid_synthase fatty acid synthases] (FAS-I and FAS-II) <ref name="FAS-II"> Bhatt, A. ''et al.'' (2007). The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development. ''Journal of Molecular Microbiology, 64(6),'' 1442-1454. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/17555433 17555433] DOI: [http://www.ncbi.nlm.nih.gov/pubmed/17555433 10.1111/j.1365-2958.2007.05761.x]</ref>. The FAS-II system prefers C16 as a starting substrate and can extend up to C56 <ref name="FAS-II system"> Marrakchi, Hedia, ''et al.'' (2000). InhA, a target of the antituberculous drug isoniazid, is involved in a mycobacterial fatty acid elongation system, FAS-II. ''Journal of Microbiology, 146,'' 289-296. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/10708367 10708367] </ref>. The FAS-II system utilizes the products from the FAS-I system as primers to extend the chain lengths further. The products of the FAS-II system are the precursors of mycolic acids. Elongation by the FAS-II system occurs by a [http://en.wikipedia.org/wiki/Condensation_reaction condensation reaction]of [http://en.wikipedia.org/wiki/Acetyl acetyl] and [http://en.wikipedia.org/wiki/Malonyl-CoA malonyl] substrates, which is achieved in three steps. Step 1 involves transfer of the acyl primer, step 2 involves [http://en.wikipedia.org/wiki/Decarboxylation decarboxylation] of the substrate to yield a [http://en.wikipedia.org/wiki/Carbanion carbanion], and step 3 involves nucleophilic attack of the carbanion to yield the elongated product <ref name="FAS-II" />.
 
[[Image:Screen_Shot_2015-04-15_at_4.07.06_PM.png|thumb|2000 px|left|Figure 1. Step 1 - Acyl Transfer  <ref name="FAS-II"/>]] [[Image:Step.png|thumb|2000 px|center|Figure 2. Step 2 - Decarboxylation  <ref name="FAS-II"/>]]
 
 
[[Image:Step_3.png|thumb|3000 px|center|Figure 3. Step 3 - Condensation  <ref name="FAS-II"/>]]
 
 
=== '''Reaction Catalyzed''' ===
 
The InhA gene encodes for the InhA protein. InhA catalyzes the NADH-dependent reduction of the ''trans'' double bond between positions C2-C3 of fatty acyl substrates. InhA prefers fatty acyl substrates of C16 or longer, which is consistent of the protein being a member of the FAS-II system. The longer chain length specificity of InhA distinguishes the enzyme from other enoyl-ACP reductase analogues.
 
[[Image:Mechanism-InhA.JPG|thumb|1000px|center|Figure 4. Reaction catalyzed by InhA protein <ref name="InhA"/> ]]
 
 
== '''General Structural Information''' ==
[[Image:Fatty Acyl Binding Crevice.jpg|thumb|250px|left|Figure 5. Fatty Acyl Binding Crevice (substrate binding loop in purple; substrates pictured inside the crevice)]]
Crystal structures of InhA reveal a <scene name='69/694241/Homotetramer_subunits_labeled/1'>homotetramer</scene> (each subunit featured with a different color) in aqueous solution with separate ligand binding sites in each subunit <ref name="InhA"> Rozwarski, D.A. ''et al.'' (1999). Crystal Structure of the ''Mycobacterium tuberculosis'' Enoyl-ACP Reductase, InhA, in Complex with NAD<sup>+</sup> and a C16 Fatty Acyl Substrate. ''Journal of Biological Chemistry, 274(22),'' 15582-15589. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/10336454 10336454] DOI: [http://www.ncbi.nlm.nih.gov/pubmed/10336454 10.1074/jbc.274.22.15582]</ref>. Each <scene name='69/694241/Monomer_fixed/1'>monomer</scene> subunit is composed of 289 residues and features a typical [http://en.wikipedia.org/wiki/Rossmann_fold Rossmann fold] (<scene name='69/694241/Rossmann_fold_better/1'>Rossmann fold</scene> - β-sheets = light blue; α-helices = dark blue) containing a single NADH binding site. The <scene name='69/694241/Secondary_structure/2'>secondary structure</scene> of InhA is made up of several α-helices (pink), β-sheets (gold), and β-turns (white/blue) <ref name="Phe149"> Bell, A.F. ''et al.''(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: [http://pubs.acs.org/doi/abs/10.1021/ja068219m 10.1021/ja068219m]</ref>. This enzyme also features a fatty acyl binding crevice that accommodates the long-chain fatty acyl substrate needed to synthesize mycolic acid precursors. The <scene name='69/694241/Helices_6and7_final/1'>α-6 and α-7 helices</scene> of the InhA form one side of the fatty acyl binding crevice, referred to as the <scene name='69/694241/Substrate_binding_loopresidues/1'>substrate binding loop</scene> (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 <ref name="InhA" />.
 
==='''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 <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|Figure 6. 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_labeled_better/1'>hydrophobic</scene> (purple) residues. The majority of these hydrophobic <scene name='69/694241/Hydrophobic_residues_elements/2'>residues</scene> 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 [[#Importance of Tyr-158|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'''===
 
[[Image:Tyr-158.jpg|thumb|250px|left|Figure 7. Tyr-158 (light blue) hydrogen bonding to the fatty acyl substrate, 2TK (red)]] One example of an amino acid that is not a part of the substrate binding loop yet interacts with the fatty acyl substrate is Tyr-158. 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 <scene name='69/694241/Tyr158_2tk/1'>Tyr-158</scene> forms the only direct hydrogen bond that exists between the InhA protein and the fatty acyl substrate. This hydrogen bond occurs between the hydroxyl 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" />. 
 
[[Image:Tyr-158_and_2TK.png|thumb|250 px|center|Figure 8. The side chain hydroxyl of Tyr-158 stabilizes the enolate intermediate of the fatty acyl substrate (2TK) <ref name=”Tyr-158”> Li, H.J. “et al.” (2014). A Structural and Energetic Model for the Slow-Onset Inhibition of the “Mycobacterium tuberculosis“ Enoyl-ACP Reductase InhA. “ACS Chemical Biology 9,” 986-993. PMID: [http://pubs.acs.org/doi/pdf/10.1021/cb400896g 24527857] </ref>.]]
== '''Catalytic Triad''' ==
 
Structural studies have shown that InhA possesses a <scene name='69/694241/Catalytic_triad_best/1'>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 <scene name='69/694241/Lys-165_nad_2oh/1'>2'-hydroxyl</scene> or <scene name='69/694241/Lys-165_nad/2'>3'-hydroxyl</scene> 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 <scene name='69/694241/Phe-149_nad/2'>catalytic Phe-149 residue in InhA</scene> 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 <scene name='69/694241/Catalytic_triad_in_structure/1'>catalytic triad</scene> 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''' ==
[[Image:Isoniazid.JPG|thumb|275px|right|Figure 9. Isoniazid Mechanism of Action <ref name="InhA"/> ]]
=== Isoniazid ===
 
Isoniazid is a first-line antibiotic that has been used to treat tuberculosis infections for over 50 years. Isoniazid is known to inhibit mycolic acid biosnthesis, which is the function of InhA. The activated form of isoniazid is covalently attached to the [http://en.wikipedia.org/wiki/Nicotinamide nicotinamide] ring of NADH. However, Isoniazid is still not an ideal antibiotic because many drug-resistant strains of tuberculosis have shown resistance to this inhibitor. Specifically, the mutation Ser-94 to Ala of InhA was sufficient enough to have isoniazid resistance <ref name="InhA" />. The mutation of Ser-94 to alanine is found around the cofactor binding site, resulting in a 20 to 160-fold reduction in affinity of NADH for the enzyme <ref name=”Ser94”> Kruh, N. “et al.” (2007). Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from ''Mycobacterium tuberculosis''. ''Protein Sci, 16(8),'' 1617-1627. PMID: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2203378/ 17600151] </ref>.
 
=== Other Inhibitors ===
 
Drug resistance of ''M. tuberculosis'' has become a huge problem for the development of antibiotics. A drug screen of potential inhibitors of InhA (300 compounds) was composed of inhibitors of the [http://en.wikipedia.org/wiki/Plasmodium_falciparum ''Plasmodium falciparum''] enoyl-reductase. The enoyl reductases of both bacteria have limited similarities; however, two compounds, CD39 and CD117, showed activity against drug-susceptible ''M. tuberculosis''. More importantly, both compounds showed activity against drug-resistant and multi-drug resistant TB. Treatment of the bacterium with the compounds resulted in the inhibition of mycolic acid and long-chain fatty acid biosynthesis, indicating that these compounds act against enzymes of both the FAS-I and FAS-II system. The benefit of the compounds having multiple targets is the reduced development of drug resistance, which is the disadvantage of isoniazid. The essential chemical groups that lead to the antimycobacterial properties of the compounds include a [http://en.wikipedia.org/wiki/Thioacetic_acid thioacetate] group and a [http://en.wikipedia.org/wiki/Butyl t-butyl] group <ref name="other inhibitors"> Vilchèze, C. ''et al.'' (2011). Novel Inhibitors of InhA Efficiently Kill Mycobacterium tuberculosis under Aerobic and Anaerobic Conditions. ''Antimicrobial Agents and Chemotherapy, 55(8),'' 3889-3898. DOI: [http://aac.asm.org/content/55/8/3889.full#T1 10.1128/AAC.00266-11]</ref>. 
 
[[Image:CD39.JPG|thumb|500px|left|Figure 10. CD39 Structure <ref name="other inhibitors"/>]] [[Image:CD117.JPG|thumb|600 px|center|Figure 11. CD117 Structure <ref name="other inhibitors"/>]]


This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.


</StructureSection>
</StructureSection>
== References ==
== References ==
<references/>
<references/>
==Student Contributors==
*Arielle Russell
*Mackenzie A. Smith
==Similar Proteopedia Pages==
[http://www.proteopedia.org/wiki/index.php/Enoyl-Acyl-Carrier_Protein_Reductase Enoyl-Acyl-Carrier Protein Reductase]
==Additional 3D Structures of Enoyl-ACP Reductase InhA==
[[3oew]], [[2x22]], [[2x23]], [[1eny]], [[1enz]], [[4dqu]], [[4dre]] - MtENR+NAD;
[[3of2]], [[4dti]] - MtENR(mutant)+NAD;
[[2pr2]], [[2idz]], [[2h9i]] - MtENR+INH-NAPD;
[[2aq8]] - MtENR+NADH;
[[2aqh]], [[2aqi]], [[2aqk]], [[3oey]] - MtENR(mutant)+NADH;
[[2ntj]] - MtENR+PTH-NAD;
[[2ie0]], [[2ieb]], [[2nv6]], [[1zid]] - MtENR(mutant)+INH-NAPD;
[[3fne]], [[3fnf]], [[3fng]], [[3fnh]], [[2b35]], [[1p45]] - MtENR+NAD+TCI;
[[2b36]], [[2b37]], [[4ohu]], [[4oim]], [[4oyr]] - MtENR+NAD+phenoxyphenol derivative;
[[2nsd]] - MtENR+NAD+piperidine derivative;
[[2h7l]], [[2h7m]], [[2h7n]], [[2h7p]], [[4u0j]], [[4tzt]], [[4tzk]], [[4trj]], [[4u0k]] - MtENR+NAD+pyrrolidine derivative;
[[4cod]], [[4bqp]], [[4bqr]], [[4bge]], [[4bii]], [[4oxk]], [[4oxn]], [[4oxy]], [[4r9r]], [[4r9s]] - MtENR+NAD + inhibitor;
[[4bgi]] - MtENR (mutant)+NAD+inhibitor;
[[1p44]] - MtENR+NAD+indole derivative;
[[1bvr]] - MtENR+NAD+fatty-acyl substrate

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA, Mackenzie A. Smith, Geoffrey C. Hoops
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