MabA (FabG1), a Mycobacterium tuberculosis protein involved in the long-chain fatty acid elongation system FAS-II

Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Université Paul Sabatier (UMR5089), 205 route de Narbonne, 31077 Toulouse cedex, France 1 Centre de Biochimie Structurale (INSERM U554 – CNRS UMR5048 – UM1), 29 rue de Navacelles, 34090 Montpellier cedex, France 2 Author for correspondence: A. Quémard. Tel: +33 5 61 17 55 76. Fax: +33 5 61 17 59 94. e-mail: annaik{at}ipbs.fr The fatty acid elongation system FAS-II is involved in the biosynthesis of mycolic acids, which are very long-chain fatty acids of the cell envelope specific to Mycobacterium tuberculosis and other mycobacteria. A potential component of FAS-II, the protein MabA (FabG1), was overexpressed and purified. Sedimentation equilibrium analyses revealed that MabA undergoes a dimer to tetramer self-association with a dissociation constant of 22 µM. The protein was detected by Western blotting in a mycobacterial cell-wall extract that produces mycolic acids and in the FPLC FAS-II fraction. MabA was shown to catalyse the NADPH-specific reduction of ß-ketoacyl derivatives, equivalent to the second step of a FAS-II elongation round. Unlike the known homologous proteins, MabA preferentially metabolizes long-chain substrates (C 8 –C 20 ) and has a poor affinity for the C 4 substrate, in agreement with FAS-II specificities. Molecular modelling of MabA structure suggested the presence of an unusually hydrophobic substrate-binding pocket holding a unique Trp residue, suitable for fluorescence spectroscopic analyses. In agreement with the enzyme kinetic data, the spectral properties of MabA were different in the presence of the C 8 –C 16 ligands as compared to the C 4 ligand. Altogether, these data bring out distinctive enzymic and structural properties of MabA, which correlate with its predilection for long-chain substrates, in contrast to most of the other known ketoacyl reductases. Keywords: ß-ketoacyl reductase, quaternary structure, enzymic activity, structural model, fluorescence spectroscopy Abbreviations: ACP, acyl carrier protein; ESI, electrospray ionization; FAS, fatty acid synthetase; H-MabA, His-tagged MabA; INH, isoniazid; MALDI-TOF, matrix-associated laser desorption ionization-time of flight; KAR, ß-ketoacyl-ACP reductase; PDB, Protein Data Bank; SDR, short-chain dehydrogenases/reductases a Present address: St Jude Children’s Research Hospital, Dept of Biochemistry, 322 N Lauderdale, Memphis, TN 38105, USA. b Hedia Marrakchi and Stéphanie Du