title: Evidence from Raman spectroscopy that InhA, the mycobacterial enoyl reductase, modulates the conformation of the NADH cofactor to promote catalysis.
abstract: InhA, the enoyl reductase from Mycobacterium tuberculosis, catalyzes the NADH-dependent reduction of trans-2-enoyl-ACPs. In the present work, Raman spectroscopy has been used to identify catalytically relevant changes in the conformation of the nicotinamide ring that occur when NADH binds to InhA. For 4(S)-NADD, there is an 11 cm-1 decrease in the wavenumber of the C4-D stretching band (nuC-D) and a 50% decrease in the width of this band upon binding to InhA. While a similar reduction in line width is observed for the corresponding band arising from 4(R)-NADD, nuC-D for this isomer increases 34 cm-1 upon binding to InhA. These changes in nuC-D indicate that the nicotinamide ring adopts a bound conformation in which the 4(S)C-D bond is in a pseudoaxial orientation. Mutagenesis of F149, a conserved active site residue close to the cofactor, demonstrates that this enzyme-induced modulation in cofactor structure is directly linked to catalysis. In contrast to the wild-type enzyme, Raman spectra of NADD bound to F149A InhA resemble those of NADD in solution. Consequently, F149A is no longer able to optimally position the cofactor for hydride transfer, which correlates with the 30-fold decrease in kcat and 2-fold increase in D(V/KNADH) caused by this mutation. These studies thus substantiate the proposal that hydride transfer is promoted by pseudoaxial positioning of the NADH pro-4S bond, and indicate that catalysis of substrate reduction by InhA results, in part, from correct orientation of the cofactor in the ground state.
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA.
authors.forename: Alasdair F
authors.initials: AF
authors.lastname: Bell
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Stratton
authors.forename: Xujie
authors.initials: X
authors.lastname: Zhang
authors.forename: Polina
authors.initials: P
authors.lastname: Novichenok
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Jaye
authors.forename: Pravin A
authors.initials: PA
authors.lastname: Nair
authors.forename: Sapan
authors.initials: S
authors.lastname: Parikh
authors.forename: Richa
authors.initials: R
authors.lastname: Rawat
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tonge
mesh_terms: D002384:Catalysis; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009243:NAD / Q000737:chemistry* / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D014732:Vibration
chemical_list: D009243:NAD; D010649:Phenylalanine; D010088:Oxidoreductases