Molecular Basis of the Functional Divergence of Fatty Acyl-AMP Ligase Biosynthetic Enzymes of Mycobacterium tuberculosis Aneesh Goyal 1 †, Priyanka Verma 2 †, Madhankumar Anandhakrishnan 1 , Rajesh S. Gokhale 2,3 ⁎‡ and Rajan Sankaranarayanan 1 ⁎ 1 Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500 007, India 2 National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India 3 Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research, Mall Road, New Delhi 110 007, India Received 14 September 2011; received in revised form 13 December 2011; accepted 14 December 2011 Available online 21 December 2011 Edited by G. Schulz Keywords: crystal structure; acyl-adenylate enzymes; pathogenesis; substrate specificity; virulent lipid synthesis Activation of fatty acids as acyl-adenylates by fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis is a variant of a classical theme that involves formation of acyl-CoA (coenzyme A) by fatty acyl-CoA ligases (FACLs). Here, we show that FAALs and FACLs possess similar structural fold and substrate specificity determinants, and the key difference is the absence of a unique insertion sequence in FACL13 structure. A systematic analysis shows a conserved hydrophobic anchorage of the insertion motif across several FAALs. Strikingly, mutagenesis of two phenylalanine residues, which are part of the anchorage, to alanine converts FAAL32 to FACL32. This insertion-based in silico analysis suggests the presence of FAAL homologues in several other non-mycobacterial genomes including eukaryotes. The work presented here establishes an elegant mechanism wherein an insertion sequence drives the functional divergence of FAALs from canonical FACLs. © 2011 Elsevier Ltd. All rights reserved. Introduction Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. Mtb houses remarkably diverse lipid repertoire that has been implicated in its virulence and pathogenesis. 1,2 Whereas the chemi- cal nature of these lipids has been known for years, studies in the last decade have provided useful insights into the novel biosynthetic machinery utilized by Mtb. All this has led to the identification of a novel enzyme family called fatty acyl-AMP ligases (FAALs). FAALs activate fatty acyl chains and transfer them to multifunctional and multi- domain enzymes called polyketide synthases (PKSs) to produce complex lipid moieties. 3 Though lipid metabolites are abundant in microbial world, the *Corresponding authors. R. S. Gokhale is to be contacted at Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research, University Campus, Mall Road, New Delhi 110 007, India; R. Sankaranarayanan, Structural Biology Laboratory, Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Habsiguda, Uppal Road, Hyderabad 500 007, India. E-mail addresses: rsg@igib.res.in; sankar@ccmb.res.in. † A.G. and P.V. contributed equally to this work. ‡ Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, India (by courtesy). Abbreviations used: FAAL, fatty acyl-AMP ligase; FACL, fatty acyl-CoA ligase; Mtb, Mycobacterium tuberculosis; PKS, polyketide synthase; AAE, acyl- activating enzyme; ACS, acetyl-CoA synthetase; PDB, Protein Data Bank; MS, mass spectrometry; DIP2C, disco-interacting protein 2 homologue C. doi:10.1016/j.jmb.2011.12.031 J. Mol. Biol. (2012) 416, 221–238 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.