Sandbox Reserved 321: Difference between revisions

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 isoniazid (INH), which have been used in treatment of mycobacterial infections <ref name ="phosphorylation of inhA">PMID:21143326</ref>.

 

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 comprised 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 of a series of α strands flanking a central β sheet of multiple parallel β strands<ref name ="crystallographic studies"/>.

 

==Hyrdrophobic Binding Pocket==

 

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 phenylalanine 41 (F41) and interactions with lysine 165 <scene name='Sandbox_Reserved_321/Lys165/1'>(K165)</scene><ref name ="Roles of T158"/><ref name ="crystallographic studies"/>.  Binding of NADH is mediated through van der Waal ineractions with the side chains of leucine 218 (K218) and methionine 155 (M155)to the phosphate group and 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 (Y158) 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 ETH-NAD adduct (EAD), and the PTH-NAD adducts (PAD) 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 with distances of 3.3Å and 3.2Å respectively<ref name ="mech of thioamide drug action"/>.  EAD or PAD 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 PAD and the ethyl group of EAD with Y158 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.

 

{*[[1bxk]] - DTDP-glucose 4,6-dehydratase -''E. coli''<br />

*[[1qsg]] - Enoyl reductase + Triclosan<br />}