Introduction

Mycobacterium Tuberculosis is an ACSVL (Acyl-CoA synthetases very long) peripheral membrane protein^[1]. ACS proteins activate lipids and fatty acids before going into metabolic pathways. FadD13 is soluble unlike other ACSVL proteins. FadD13 contains a hydrophobic tunnel for fatty acids to bind to, as well as an arginine rich lid loop that binds to the cell membrane. The binding of ATP causes structural changes promoting the binding of the hydrophobic substrates. Formation of an acyl-adenylate intermediate induces a 140 degree rotation of the small domain and binding of CoA for production of the final product, a fatty acyl-CoA thioester^[2]. Shown below is the general mechanism for ACS proteins.

Figure 1 shows the general outline of the binding of ATP and acyl substrates to an ACSVL enzyme. This is the accepted mechanism for these types of proteins.

Background

Mycobacterium Tuberculosis is the causative agent of Tuberculosis commonly abbreviated TB. TB causes approximately 1.4 million deaths every year. The cost for treatment of patients with TB between the years 2010-2015 was approximately 16 billion dollars. TB is spread through the air, not by contact. There are two forms of TB, latent TB and TB disease.

Structural Highlights

FadD13 is composed of 503 amino acids which are divided into two domains. The larger of the two domains is the N-terminal domain composed of shown in blue. The smaller of the two domains is the C-terminal domain composed of shown in yellow. These two domains are held together by a flexible 6 amino acid linker () shown in black.

Active Site

The active site of FadD13 is composed of an . This region is comprised of residues 164-TSGTTGHPKG173-173 shown in red which binds to the phosphate group, and residues 298-VQGYALTE-305 shown in blue which binds to the adenine group^[3]. Upon binding of ATP/AMP, FadD13 becomes activated and allows for binding of lipids or fatty acids.

Hydrophobic Tunnel

FadD13 has a distinct hydrophobic tunnel that starts at the active site and is capped by the positively charged surface patch.The is found inside the N-terminal domain. It is composed of six beta sheets (beta 9-14) shown in green and two alpha helices (alpha 8-9) shown in red.The hydrophobic tunnel allows large lipids/fatty acids, up to 26 carbons, to bind.

Surface Patch

FadD13 has an arginine and aromatic rich surface patch that allows it to be a peripheral-membrane protein^[4].

Figure 2 shows the proposed mechanism for an ACSVL protein bound to the membrane^[5]

Function

The FadD13 enzyme functions to activate lipids. Once the lipids are activated, they can continue on into metabolic pathways. This is done by ATP/AMP binding to the . Once ATP/AMP is bound, the long lipid chain up to 26 carbons may bind in the of the enzyme. Upon binding of the substrate, the C terminal swings up to close off the tunnel. From there CoA can bind to produce the final product, an acyl-CoA Thioester. The lipid can now move transversely throughout the membrane and throughout the rest of the cell. Below is the proposed mechanism for ACSVL proteins.