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Summer Research Fellowship Programme of India's Science Academies

Analysis of the mode of substrate binding in Fatty acyl adenylate ligases (FAAL) and Fatty acyl CoA ligases (FACL)

Abhinav Bharathwaj Swaminathan

Department of Biotechnology, Alagappa College of Technology, Anna University, Chennai-600025.

Dr. Rajan Sankaranarayanan

CSIR-Centre for Cellular and Molecular Biology (CCMB), Habsiguda, Hyderabad, Telengana -500007

Abstract

The survival of an organism depends on two major classes of chemical reactions, namely catabolic and anabolic reactions. Lipid metabolism in prokaryotes is channelled through these two distinct pathways by enzymes called fatty acyl-CoA ligases (FACLs) and fatty acyl-AMP ligases (FAALs). Fatty acyl-AMP ligases can act as stand-alone proteins or fused to multi-domain proteins such as Polyketide synthases (PKSs) and Non-ribosomal peptide synthetase-Polyketide synthase hybrids (NRPS-PKSs) for delivering activated fatty acids for production of complex metabolites. Such an adenylating function followed by fatty acyl transfer to the phosphopantetheine arm of Acyl carrier protein (ACP) is also found in other PKSs and Non-ribosomal peptide synthetases (NRPS), whose adenylation domain (A-domains) share structural homology with FAALs and FACLs. Previous studies from the lab suggest that FAALs differs from FACLs and A-domains of NRPS as they fail to react with Coenzyme-A(CoASH). Further characterization is required for a holistic understanding of the mechanistic principles that ensure the peculiar biochemical property of FAALs. Therefore, we cloned FAAL and Adenylation domain-ACP di-domain constructs from Bacillus subtilis, which will be used as model system for further experimentation. These constructs of FAAL and Adenylation domain-ACP di-domain can help in the process of crystallization of FAAL and its ACP in its thioester-forming conformation along with the amenability to perform biochemical analysis through structure-guided mutagenesis. In silico analysis of the substrate-binding pocket of these enzymes was performed and compared with well characterized FAALs and FACLs to predict the potential substrate of these enzymes.

Keywords: Acyl-adenylate enzymes, Lipid biosynthesis, Lipid catabolism.

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