title: Role of Serine140 in the mode of action of Mycobacterium tuberculosis β-ketoacyl-ACP Reductase (MabA).
abstract: BACKGROUND
  Tuberculosis (TB) still remains one of the most deadly infectious diseases in the world. Mycobacterium tuberculosis β-ketoacyl-ACP Reductase (MabA) is a member of the fatty acid elongation system type II, providing precursors of mycolic acids that are essential to the bacterial cell growth and survival. MabA has been shown to be essential for M. tuberculosis survival and to play a role in intracellular signal transduction of bacilli.
  
  
  RESULTS
  Here we describe site-directed mutagenesis, recombinant protein expression and purification, steady-state kinetics, fluorescence spectroscopy, and molecular modeling for S140T and S140A mutant MabA enzymes. No enzyme activity could be detected for S140T and S140A. Although the S140T protein showed impaired NADPH binding, the S140A mutant could bind to NADPH. Computational predictions for NADPH binding affinity to WT, S140T and S140A MabA proteins were consistent with fluorescence spectroscopy data.
  
  
  CONCLUSIONS
  The results suggest that the main role of the S140 side chain of MabA is in catalysis. The S140 side chain appears to also play an indirect role in NADPH binding. Interestingly, NADPH titrations curves shifted from sigmoidal for WT to hyperbolic for S140A, suggesting that the S140 residue may play a role in displacing the pre-existing equilibrium between two forms of MabA in solution. The results here reported provide a better understanding of the mode of action of MabA that should be useful to guide the rational (function-based) design of inhibitors of MabA enzyme activity which, hopefully, could be used as lead compounds with anti-TB action.
authors.affiliation: Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, Porto Alegre, RS 90619-900, Brazil.
authors.forename: Leonardo A
authors.initials: LA
authors.lastname: Rosado
authors.forename: Rafael Andrade
authors.initials: RA
authors.lastname: Caceres
authors.forename: Walter Filgueira
authors.initials: WF
authors.lastname: de Azevedo
authors.forename: Luiz A
authors.initials: LA
authors.lastname: Basso
authors.forename: Diógenes S
authors.initials: DS
authors.lastname: Santos
mesh_terms: D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012694:Serine; D009249:NADP; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase