Mycobacterium tuberculosis -Ketoacyl-Acyl Carrier Protein (ACP) Reductase: Kinetic and Chemical Mechanisms ² Rafael G. Silva, ‡,§ Luiz Pedro S. de Carvalho, ‡,| John S. Blanchard, ⊥ Dio ´genes S. Santos,* ,‡ and Luiz A. Basso* ,‡ Centro de Pesquisas em Biologia Molecular e Funcional, Faculdade de Biocie ˆ ncias e Faculdade de Farma ´ cia, Instituto de Pesquisas Biome ´ dicas, PUCRS, 6681/92-A AVenida Ipiranga, 90619-900, Porto Alegre, RS, Brazil, Programa de Po ´ s-graduac ¸ a ˜ o em Cie ˆ ncias Biolo ´ gicas: Bioquı ´mica, Instituto de Cie ˆ ncias Ba ´ sicas da Sau ´ de, UniVersidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil, and Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park AVenue, Bronx, New York 10461 ReceiVed June 6, 2006; ReVised Manuscript ReceiVed August 11, 2006 ABSTRACT: -ketoacyl-acyl carrier protein (ACP) reductase from Mycobacterium tuberculosis (MabA) is responsible for the second step of the type-II fatty acid elongation system of bacteria, plants, and apicomplexan organisms, catalyzing the NADPH-dependent reduction of -ketoacyl-ACP to generate -hydroxyacyl-ACP and NADP + . In the present work, the mabA-encoded MabA has been cloned, expressed, and purified to homogeneity. Initial velocity studies, product inhibition, and primary deuterium kinetic isotope effects suggested a steady-state random bi-bi kinetic mechanism for the MabA-catalyzed reaction. The magnitudes of the primary deuterium kinetic isotope effect indicated that the C 4 -proS hydrogen is transferred from the pyridine nucleotide and that this transfer contributes modestly to the rate-limiting step of the reaction. The pH-rate profiles demonstrated groups with pK values of 6.9 and 8.0, important for binding of NADPH, and with pK values of 8.8 and 9.6, important for binding of AcAcCoA and for catalysis, respectively. Temperature studies were employed to determine the activation energy of the reaction. Solvent kinetic isotope effects and proton inventory analysis established that a single proton is transferred in a partially rate-limiting step and that the mechanism of carbonyl reduction is probably concerted. The observation of an inverse D 2 O V/K and an increase in D 2 O V when [4S- 2 H]NADPH was the varied substrate obscured the distinction between stepwise and concerted mechanisms; however, the latter was further supported by the pH dependence of the primary deuterium kinetic isotope effect. Kinetic and chemical mechanisms for the MabA-catalyzed reaction are proposed on the basis of the experimental data. Tuberculosis (TB), 1 an infectious disease caused by the Gram-positive bacillus Mycobacterium tuberculosis, is a major human health threat, with an annual rate of 8.8 million new cases and 1.7 million deaths, 95% of which occur in developing countries (1). The treatment of TB demands a standard 6 month administration of several drugs, whose side effects lead to patient noncompliance (2). In addition, there is an increasing incidence of infection with multidrug- resistant tuberculosis strains (MDR-TB), defined as being resistant to at least two drugs commonly used to treat TB, isoniazid and rifampicin (3). There is thus an urgent need for the development of new antimycobacterial agents, and the detailed molecular characterization of new targets is a step of paramount importance toward this goal. The type-II fatty acid elongation system (FAS II) of bacteria, in which the reactions are catalyzed by different enzymes, each encoded by a discrete gene, constitutes an attractive target for inhibition, because it contrasts with the type-I fatty acid elongation system (FAS I) of mammals, whose reactions are catalyzed by a single, multifunctional polypeptide (4). M. tuberculosis possesses both systems. FAS I is responsible for the biosynthesis of acyl chains with 14- 16 carbons, while FAS II generates fatty acids with 25-56 carbons. The two systems provide precursors for the bio- synthesis of mycolic acids, which contain very long-chain fatty acids that are prominent and essential components of the mycobacterial cell wall (5). The FAS II system of mycobacteria elongates fatty acids by adding C2 units to the ² This work was supported by Millennium Initiative Program MCT- CNPq, Ministry of Health/DECIT-Secretary of Health Policy, and PRONEX/FAPERGS/CNPq (Brazil) to D.S.S. and L.A.B. and NIH Grant AI33696 to J.S.B. D.S.S. (304051/1975-06) and L.A.B. (520182/ 99-5) are Research Career Awardees from the National Research Council of Brazil (CNPq). R.G.S. is a predoctoral fellow from CNPq. * To whom correspondence should be addressed: Telephone/Fax: +55-51-33203629. E-mail: luiz.basso@pucrs.br (L.A.B.); diogenes@ pucrs.br (D.S.S.). ‡ Instituto de Pesquisas Biome ´dicas. § Instituto de Cie ˆncias Ba ´sicas da Sau ´de. | Current address: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. ⊥ Albert Einstein College of Medicine. 1 Abbreviations: TB, tuberculosis; MDR-TB, multidrug-resistant tuberculosis; FAS II, type-II fatty acid elongation system; FAS I, type-I fatty acid elongation system; ACP, acyl carrier protein; MabA, -ketoacyl-ACP reductase from M. tuberculosis, FabG, -ketoacyl- ACP reductase from other organisms; SDR, short-chain dehydrogenases/ reductases; AcAcCoA, acetoacetyl-coenzyme A; -HBCoA, -hy- droxyacetyl-coenzyme A, IPTG, isopropyl-1-thio--D-galactopyranoside; NADPD, deuterated pyridine nucleotide; ESI-MS, electronspray ionization mass spectrometry; InhA, 2-trans-enoyl-ACP reductase from M. tuberculosis. 13064 Biochemistry 2006, 45, 13064-13073 10.1021/bi0611210 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/07/2006