Sandbox Reserved 321: Difference between revisions

Line 42:

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>.

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 ~~mediates~~ binding<ref name ="Roles of T158"/><ref name ="crystallographic studies"/>. Binding og 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 ~~NAD+~~<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:

The primary target of the thioamide drugs PTH and ETH ~~has~~ been shown to be InhA <scene name='Sandbox_Reserved_321/Structural_progresion/1'>(go to original scene)</scene> in both ~~gentic~~ 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 ~~vivo~~]<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 it<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"/>. ~~Curently~~ studies are being carried out to determine other methods of treatment for mycobaterial infections that dont require activation by cellular constituents, due to the ~~incereasing in~~ 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>, and the PTH-NAD adducts (P1H) have been found to occupy the same hydrophobic pocket of InhA as NADH and exhibit the same van der Waal interactions between K218 and M155 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 rotaion of F149 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. If InhA is no longer active then the mycolic acids nessasary in cell wall compostion of various mycobacteria will not be formed.

Sandbox Reserved 321: Difference between revisions

Navigation menu

Page actions

Page actions

Personal tools

Navigation

Search

Tools

@@ Line 42: / Line 42: @@
 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>.
-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 mediates binding<ref name ="Roles of T158"/><ref name ="crystallographic studies"/>.   Binding og 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 NAD+<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 50: @@
 [[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]]
-The primary target of the thioamide drugs PTH and ETH has been shown to be InhA <scene name='Sandbox_Reserved_321/Structural_progresion/1'>(go to original scene)</scene> in both gentic 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 vivo]<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 it<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"/>.  Curently studies are being carried out to determine other methods of treatment for mycobaterial infections that dont require activation by cellular constituents, due to the incereasing in 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>, and the PTH-NAD adducts (P1H) have been found to occupy the same hydrophobic pocket of InhA as NADH and exhibit the same van der Waal interactions between K218 and M155 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 rotaion of F149 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.  If InhA is no longer active then the mycolic acids nessasary in cell wall compostion of various mycobacteria will not be formed.