Sandbox Reserved 321: Difference between revisions
No edit summary |
No edit summary |
||
| (2 intermediate revisions by the same user not shown) | |||
| Line 8: | Line 8: | ||
by Kelly Hrywkiw | by Kelly Hrywkiw | ||
{{STRUCTURE_2h9i | PDB=2h9i | SCENE= }} | {{STRUCTURE_2h9i | PDB=2h9i | SCENE= }} | ||
__TOC__ | __TOC__ | ||
| Line 15: | Line 14: | ||
=Introduction= | =Introduction= | ||
The enzyme InhA is coded from the INHA gene that is similar in sequence to the ''[http://en.wikipedia.org/wiki/Salmonella_typhimurium Salmonella typhimurium]''gene which plays a role in [http://en.wikipedia.org/wiki/Fatty_acid_synthesis fatty acid synthesis], and is part of a short chain dehydrogenase/reductase family<ref name ="making drugs for inhA">Sacchettini, James (New Rochelle, NY) 1999 INHA crystals and three dimensional structure United States Albert Einstein College of Medicine of Yeshiva University (Bronx, NY) 5882878 http://www.freepatentsonline.com/5882878.html</ref><ref name ="phosphorylation of inhA">PMID:21143326</ref>. Inha is an [http://en.wikipedia.org/wiki/NADH NADH] dependent trans enoyl-acyl ACP carrier protein that is part of the fatty acid biosynthesis system: fatty acid synthase two (FASII), and plays a role in the synthesis of [http://en.wikipedia.org/wiki/Mycolic_acid Mycolic Acid]<ref name ="mech of thioamide drug action">PMID:17227913</ref><ref name ="phosphorylation of inhA">PMID:21143326</ref>. Mycolic acids are long chain fatty acids (C54 to C63) that are essential in cell wall formation of the human pathogen ''[http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis Mycobacterium tuberculosis]''as well as other mycobateria such as ''[http://en.wikipedia.org/wiki/Mycobacterium_leprae Mycobacterium leprae]'', and are associated with virulence<ref name ="TB">PMID2568869:</ref>. InhA has been proposed as the target of the [http://en.wikipedia.org/wiki/Thioamidedrugs thioamide] drugs, ethionamide (ETH) and protionamide (PTH), which have been used in treatment of mycobacterial infections <ref name ="phosphorylation of inhA">PMID:21143326</ref>. However stains of ''M. tuberculosis that are resistant to thioamide drugs have been increaseing worldwide, and therefor research into the exact mechanisms of these drugs is of importance. | The enzyme InhA is coded from the INHA gene that is similar in sequence to the ''[http://en.wikipedia.org/wiki/Salmonella_typhimurium Salmonella typhimurium]''gene which plays a role in [http://en.wikipedia.org/wiki/Fatty_acid_synthesis fatty acid synthesis], and is part of a short chain dehydrogenase/reductase family<ref name ="making drugs for inhA">Sacchettini, James (New Rochelle, NY) 1999 INHA crystals and three dimensional structure United States Albert Einstein College of Medicine of Yeshiva University (Bronx, NY) 5882878 http://www.freepatentsonline.com/5882878.html</ref><ref name ="phosphorylation of inhA">PMID:21143326</ref>. Inha is an [http://en.wikipedia.org/wiki/NADH NADH] dependent trans enoyl-acyl ACP carrier protein that is part of the fatty acid biosynthesis system: fatty acid synthase two (FASII), and plays a role in the synthesis of [http://en.wikipedia.org/wiki/Mycolic_acid Mycolic Acid]<ref name ="mech of thioamide drug action">PMID:17227913</ref><ref name ="phosphorylation of inhA">PMID:21143326</ref>. Mycolic acids are long chain fatty acids (C54 to C63) that are essential in cell wall formation of the human pathogen ''[http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis Mycobacterium tuberculosis]''as well as other mycobateria such as ''[http://en.wikipedia.org/wiki/Mycobacterium_leprae Mycobacterium leprae]'', and are associated with virulence<ref name ="TB">PMID2568869:</ref>. InhA has been proposed as the target of the [http://en.wikipedia.org/wiki/Thioamidedrugs thioamide] drugs, ethionamide (ETH) and protionamide (PTH), which have been used in treatment of mycobacterial infections <ref name ="phosphorylation of inhA">PMID:21143326</ref>. However stains of ''M. tuberculosis'' that are resistant to thioamide drugs have been increaseing worldwide, and therefor research into the exact mechanisms of these drugs is of importance. | ||
=Structure of InhA= | =Structure of InhA= | ||
[[Image:Stero veiw.png|thumb|right|upright=2.5|alt=Secondary Structure Succession of InhA. Secondary structure residues are ordered from blue to red.|Fig.1: Stero view of the homotetramer structure of InhA with secondary structure succession outlined]] | |||
<Structure load='2h9i' size='275' frame='true' align='left' caption='Momomeric subunit of InhA with bound EAD' scene='Sandbox_Reserved_321/Structural_progresion/1' /> | <Structure load='2h9i' size='275' frame='true' align='left' caption='Momomeric subunit of InhA with bound EAD' scene='Sandbox_Reserved_321/Structural_progresion/1' /> | ||
The InhA enzyme <scene name='Sandbox_Reserved_321/Structural_progresion/1'>(go to original scene)</scene> of ''M. tuberculosis'' is a homotetramer composed of a repeating subunit of a single domain with a [http://en.wikipedia.org/wiki/Rossmann_fold Rossmann Fold] in the core that provides a NADH binding site<ref name ="crystallographic studies"/>. The single domain can be broken down into two substructures that are connected by short peptide loop<ref name ="making drugs for inhA"/><ref name ="crystallographic studies">PMID:17588773</ref>. The overall structure exhibits α/β folding | The InhA enzyme <scene name='Sandbox_Reserved_321/Structural_progresion/1'>(go to original scene)</scene> of ''M. tuberculosis'' is a homotetramer (Fig. 1) composed of a repeating subunit of a single domain with a [http://en.wikipedia.org/wiki/Rossmann_fold Rossmann Fold] in the core that provides a NADH binding site<ref name ="crystallographic studies"/>. The single domain can be broken down into two substructures that are connected by short peptide loop<ref name ="making drugs for inhA"/><ref name ="crystallographic studies">PMID:17588773</ref>. The overall structure exhibits α/β folding with a series of [http://en.wikipedia.org/wiki/Alpha_helix α helices] flanking a central [http://en.wikipedia.org/wiki/Beta_sheet β sheet] of multiple parallel β strands<ref name ="crystallographic studies"/>. | ||
==Substructure 1 of InhA== | ==Substructure 1 of InhA== | ||
| Line 40: | Line 41: | ||
=InhA's Function in the Mycolic Acid Pathway= | =InhA's Function in the Mycolic Acid Pathway= | ||
[[Image:Pathway2.png|thumb|right|upright=2|alt=Proposed mechanism.|Formulated mechanism of Mycolic acid synthesis as proposed by Wilson et al.<ref name ="Drug Induced Alterations">PMID:10536008</ref>.]] | [[Image:Pathway2.png|thumb|right|upright=2|alt=Proposed mechanism.|Fig 2: Formulated mechanism of Mycolic acid synthesis as proposed by Wilson et al.<ref name ="Drug Induced Alterations">PMID:10536008</ref>.]] | ||
InhA plays a key role in the synthesis of fatty acids, particularly in ''M. tuberculosis'' which, has type one fatty acid synthesis (FASI) and type two fatty acid synthesis (FASII) which together function in the synthesis of mycolic acids<ref name ="Function of M Tb">PMID:18552191</ref>. FASI synthesizes C16-18 and C24-26 fatty acids. The fatty acids from FASI are then sent to FASII which promotes chain extension, forming long-chain meromycolic acids that are 56-64 carbons in length<ref name ="Fatty Acid Synthesis">PMID:18804030</ref>. The final step in FASII is completed by InhA which reduces 2-trans-enoyl-ACP's with chain lengths over twelve carbons in a NADP dependent manner where the hydride transfer precedes protonation<ref name ="Function of M Tb"/><ref name ="Roles of T158">PMID:10521269</ref>. | InhA plays a key role in the synthesis of fatty acids, particularly in ''M. tuberculosis'' which, has type one fatty acid synthesis (FASI) and type two fatty acid synthesis (FASII) which together function in the synthesis of mycolic acids<ref name ="Function of M Tb">PMID:18552191</ref>. FASI synthesizes C16-18 and C24-26 fatty acids. The fatty acids from FASI are then sent to FASII which promotes chain extension, forming long-chain meromycolic acids that are 56-64 carbons in length<ref name ="Fatty Acid Synthesis">PMID:18804030</ref>. The final step in FASII is completed by InhA which reduces 2-trans-enoyl-ACP's with chain lengths over twelve carbons in a NADP dependent manner where the hydride transfer precedes protonation(Fig. 2)<ref name ="Function of M Tb"/><ref name ="Roles of T158">PMID:10521269</ref>. | ||
The reaction takes place as follows: initially NADH binds to the active site mediated by [http://en.wikipedia.org/wiki/Van_der_Waals_force van der Waal] interactions with the side chains of phenylalanine 41 (F41), leucine 218 and methionine 155 <scene name='Sandbox_Reserved_321/K218_and_m_155/1'>(K218 and M155)</scene> to the phosphate group of NADH. There are additional interaction with lysine 165 <scene name='Sandbox_Reserved_321/Lys165/1'>(K165)</scene> that also mediate binding<ref name ="Roles of T158"/><ref name ="crystallographic studies"/>. Binding of NADH causes a conformational change in the Aspartate 42 and Arginine 43 <scene name='Sandbox_Reserved_321/Asp_42_and_arg_43/1'>(E42 and R43)</scene> side chains and an over all conformational change in InhA<ref name ="crystallographic studies"/><ref name ="mech of thioamide drug action"/>. In addition tyrosine 158 <scene name='Sandbox_Reserved_321/Tyr_158/1'>(Y158)</scene> plays an important role in aligning the carbonyl substrate, in fact; rotation about its Cα-Cβ bond by 60° brings it into a position where it can hydrogen bond to the carbonyl of the 2-trans enoyl-ACP and provide it with electrophilic stabilization<ref name ="Roles of T158"/>. The substrate binds in a U-shaped conformation with its trans double bond adjacent to the nicotinamide ring of NADH<ref name ="Fatty acyl in InhA"/>. Inha then reduces the 2-trans double bond of the substrate by forming a enoyl intermediate through the transfer of a hydride ion from NADH to the third carbon of the substrate, followed by protonation of the second carbon<ref name ="crystallographic studies"/>. The binding of both the substrate and the cofactor induces another conformational change in InhA that allows for the release of the meromycolic acid product<ref name ="crystallographic studies"/>. The meromycolic acids undergo [http://en.wikipedia.org/wiki/Claisen_condensation claisen condensation] with a C26 fatty acid followed by reduction to a mature mycolic acid<ref name ="Fatty Acid Synthesis"/><ref name ="crystallographic studies"/>. | The reaction takes place as follows: initially NADH binds to the active site mediated by [http://en.wikipedia.org/wiki/Van_der_Waals_force van der Waal] interactions with the side chains of phenylalanine 41 (F41), leucine 218 and methionine 155 <scene name='Sandbox_Reserved_321/K218_and_m_155/1'>(K218 and M155)</scene> to the phosphate group of NADH. There are additional interaction with lysine 165 <scene name='Sandbox_Reserved_321/Lys165/1'>(K165)</scene> that also mediate binding<ref name ="Roles of T158"/><ref name ="crystallographic studies"/>. Binding of NADH causes a conformational change in the Aspartate 42 and Arginine 43 <scene name='Sandbox_Reserved_321/Asp_42_and_arg_43/1'>(E42 and R43)</scene> side chains and an over all conformational change in InhA<ref name ="crystallographic studies"/><ref name ="mech of thioamide drug action"/>. In addition tyrosine 158 <scene name='Sandbox_Reserved_321/Tyr_158/1'>(Y158)</scene> plays an important role in aligning the carbonyl substrate, in fact; rotation about its Cα-Cβ bond by 60° brings it into a position where it can hydrogen bond to the carbonyl of the 2-trans enoyl-ACP and provide it with electrophilic stabilization<ref name ="Roles of T158"/>. The substrate binds in a U-shaped conformation with its trans double bond adjacent to the nicotinamide ring of NADH<ref name ="Fatty acyl in InhA"/>. Inha then reduces the 2-trans double bond of the substrate by forming a enoyl intermediate through the transfer of a hydride ion from NADH to the third carbon of the substrate, followed by protonation of the second carbon<ref name ="crystallographic studies"/>. The binding of both the substrate and the cofactor induces another conformational change in InhA that allows for the release of the meromycolic acid product<ref name ="crystallographic studies"/>. The meromycolic acids undergo [http://en.wikipedia.org/wiki/Claisen_condensation claisen condensation] with a C26 fatty acid followed by reduction to a mature mycolic acid<ref name ="Fatty Acid Synthesis"/><ref name ="crystallographic studies"/>. | ||
| Line 50: | Line 51: | ||
<Structure load='2h9i' size='275' frame='true' align='left' caption='Momomeric subunit of InhA with bound EAD' scene='Sandbox_Reserved_321/Structural_progresion/1' /> | <Structure load='2h9i' size='275' frame='true' align='left' caption='Momomeric subunit of InhA with bound EAD' scene='Sandbox_Reserved_321/Structural_progresion/1' /> | ||
[[Image:ETH, EAD, PTH, P1H structures.png|thumb|right|upright=1.5|alt=ETH, EAD, PTH, and P1H.|Structures of ETH, EAD, PTH, and P1H]] | [[Image:ETH, EAD, PTH, P1H structures.png|thumb|right|upright=1.5|alt=ETH, EAD, PTH, and P1H.|Fig 3: Structures of ETH, EAD, PTH, and P1H]] | ||
The primary target of the thioamide drugs PTH and ETH have been shown to be InhA <scene name='Sandbox_Reserved_321/Structural_progresion/1'>(go to original scene)</scene> in both genetic and molecular experiments<ref name ="mech of thioamide drug action"/>. Both PTH and ETH require activation by various cellular componets to form the NAD adduct that acts to inhibit InhA, and therefore connot be studied in [http://en.wikipedia.org/wiki/In_vitro in vitro]<ref name ="mech of thioamide drug action"/>. The exacct mechanism of their activation is still under speculation, however a flavin monooxygenase (EthA) has been shown to participate in ETH and PTH activation<ref name ="mech of thioamide drug action"/>. In fact, strains of ''M. tuberculosis'' that have mutations in the gene which express EthA exhibit resistance to thioamide drugs<ref name ="mech of thioamide drug action"/>. Currently studies are being carried out to determine other methods of treatment for mycobaterial infections that dont require activation by cellular constituents, due to the incerease of drug resistant cases world wide. | The primary target of the thioamide drugs PTH and ETH have been shown to be InhA <scene name='Sandbox_Reserved_321/Structural_progresion/1'>(go to original scene)</scene> in both genetic and molecular experiments<ref name ="mech of thioamide drug action"/>. Both PTH and ETH require activation by various cellular componets to form the NAD adduct that acts to inhibit InhA, and therefore connot be studied in [http://en.wikipedia.org/wiki/In_vitro in vitro]<ref name ="mech of thioamide drug action"/>. The exacct mechanism of their activation is still under speculation, however a flavin monooxygenase (EthA) has been shown to participate in ETH and PTH activation<ref name ="mech of thioamide drug action"/>. In fact, strains of ''M. tuberculosis'' that have mutations in the gene which express EthA exhibit resistance to thioamide drugs<ref name ="mech of thioamide drug action"/>. Currently studies are being carried out to determine other methods of treatment for mycobaterial infections that dont require activation by cellular constituents, due to the incerease of drug resistant cases world wide. | ||
The ETH-NAD adduct <scene name='Sandbox_Reserved_321/Ligand/1'>(EAD)</scene> | The ETH-NAD adduct <scene name='Sandbox_Reserved_321/Ligand/1'>(EAD)</scene> and the PTH-NAD adduct (P1H) have been found to occupy the same hydrophobic pocket of InhA as NADH and exhibit the same van der Waal interactions between <scene name='Sandbox_Reserved_321/K218_and_m_155/1'>(K218 and M155)</scene> and the ethyl or proply group with distances of 3.3Å and 3.2Å respectively<ref name ="mech of thioamide drug action"/>. EAD or P1H binding forces the rotation of <scene name='Sandbox_Reserved_321/Phe_149/1'>F149</scene> by 90° which causes a ring stacking interation with the pyridine ring on the adduct. In addtion π stacking interactions form between the propyl group of P1H and the ethyl group of <scene name='Sandbox_Reserved_321/Pi_stacking/1'>EAD with Y158</scene> at distance of ~3.3Å. These interations and conformational changes in InhA contribute to its inactivation. Developmentaly this is important, for InhA is no longer active an the mycolic acids nessasary in cell wall compostion of various mycobacteria will not be formed. | ||