Biochem. J. (2013) 450, 127–139 (Printed in Great Britain) doi:10.1042/BJ20121107 127 Crystal structure of hexanoyl-CoA bound to β -ketoacyl reductase FabG4 of Mycobacterium tuberculosis Debajyoti DUTTA 1 , Sudipta BHATTACHARYYA, Amlan ROYCHOWDHURY, Rupam BISWAS and Amit Kumar DAS 2 Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India FabGs, or β -oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β -oxoacyl-ACP (acyl-carrier protein) into β - hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxin- type’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β -oxoacyl-CoA to β -hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD + –hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C 16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg 146 and Arg 445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis. Key words: co-operativity, hexanoyl-CoA, Mycobacterium tuberculosis, short-chain dehydrogenase/reductase, β -oxoacyl reductase. INTRODUCTION Type II fatty acid metabolism in Mycobacterium tuberculosis is particularly important for the synthesis of very-long-chain fatty acids [1]. Very-long-chain fatty acids are further processed to yield a meromycolate backbone and are ultimately destined for mycolic acid synthesis. The enzyme FabG is committed to the second step of type II fatty acid synthesis where NADPH is used to reduce β - oxoacyl-ACP (acyl-carrier protein) to β -hydroxyacyl-ACP. The M. tuberculosis genome consists of multiple FabG genes [2], two out of five of which are conserved among mycobacterial species. FabG1 (MabA) is associated with type II fatty acid metabolism [3], whereas FabG4 in mycobacteria belongs to an operon possibly involved in a non-conventional processing of fatty acids [4,5]. McFadden and co-workers have shown that FabG4 is essential for mycobacterial growth in Roisin’s minimal medium [6]. Functional complementation in yeast reveals that FabG4 can participate in the fatty acid biosynthetic pathway [7]. FabG4 is expressed in Sauton’s medium with a sub-inhibitory concentration of streptomycin [8] and hence it may play a role in drug resistance in mycobacterial species. HMwFaG (high-molecular-weight FabG) is a distinct group of β -oxoacyl reductases mostly found in bacteria including Actin- obacteria and certain lipid rich Proteobacteria. Mycobacterial FabG4 belongs to the HMwFabG group. FabG1, in contrast, belongs to the LMwFabG (low-molecular-mass FabG) group. There are two apparent differences between these two groups. First, HMwFabGs possess an N-terminal domain containing a flavodoxin-type fold. This N-terminal domain is absent in LMwFabGs. In HMwFabGs the domain is primarily responsible for stable oligomeric assembly [9]. Secondly, the C-terminal catalytic domain of HMwFabG requires NADH as hydride donor. The C-terminal domain is responsible for β -oxoacyl reductase activity which is common to both HMwFabGs and LMwFabGs, such as EcFabG (Escherichia coli FabG) and MtFabG1 (M. tuberculosis FabG1), but the LMwFabGs require NADPH for catalysis. Although FabG4 requires NADH for its β -oxoacyl-CoA reductase activity [7,9], it does not share any structural similarity or sequence homology with the NAD + -utilizing 3-hydroxyacyl- CoA dehydrogenase that is required for the β -oxidation pathway [10]. FabG is homologous to the enoyl reductase (FabI) and the only ternary complex structure of a reductase of fatty acid metabolic pathway is mycobacterial FabI [InhA (enoyl-ACP reductase)] complexed with NAD + and C 16 fattyacyl N-acetylcysteamine substrates (PDB code 1BVR [11]). Although FabG and FabI are structurally homologous they are distinguishable in several aspects like sequence similarity, substrate specificity, active-site architecture and length of the α-helix in their subdomain [3,12,13]. Both FabG and FabI accept fattyacyl substrates covalently linked with either CoA or the phosphopantetheine moiety of ACP. Owing to the lack of a whole phosphopantetheine moiety in the C 16 fattyacyl substrate of the InhA–substrate complex structure, the interaction Abbreviations used: ACP, acyl-carrier protein; EcFabG, Escherichia coli FabG; HMwFaG, high-molecular-weight FabG; InhA, enoyl-ACP reductase; LB, Luria–Bertani; LMwFabG, low-molecular-mass FabG; Mt FabG1, Mycobacterium tuberculosis FabG1; FAS, fatty-acid synthase; Ni-NTA, Ni 2 + - nitrilotriacetate; r.m.s.d., root mean square deviation; SaFabG1, Staphylococcus aureus FabG1. 1 Present address: Children’s Hospital, Harvard Medical School, CLSB 3rd Floor, 3 Blackfan Circle, Boston, MA 02115,U.S.A. 2 To whom correspondence should be addressed (email amitk@hijli.iitkgp.ernet.in). The structure factor files and co-ordinates of the FabG4 binary and ternary complexes are deposited in the PDB under the accession codes 4FW8 and 3V1U. c  The Authors Journal compilation c  2013 Biochemical Society Biochemical Journal www.biochemj.org