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.^[3]

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^[3]. Research has also shown that altering V209D, D382A, and W377A effects the structual stability of FadD13. and showed marginally reduced cytoplasmic expression, while showed a noteworthy low cytosolic expression^[4].

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. Research has also shown that plays a major role in binding of CoA. Ser 404 was shown to have a 4-fold enhancement for the Km value of CoA.^[4]

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].The hydrophobic tunnel is capped by the arginine and aromatic rich shown in yellow that is involved in the peripheral binding of the enzyme to the membrane. Based on the structural information present and biochemical information, it is likely that the lid loop opens up upon contact with the membrane. This would allow for the substrate to bind and have the lipid tail to reside in the membrane during catalysis (figure 2)^[3]. Six key arginine residues, create a positively charged surface that is likely involved in initially recruiting FadD13 to the membrane. When these residues were replaced with hyrdrophobic alanine residues, membrane binding increased. This points to the important role of hydrophobic interactions in keeping the protein bound at the membrane^[3].

Figure 2 shows the proposed mechanism for an ACSVL protein bound to the membrane^[3]

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.

Future Work

Relevant Pages

• 3T5B
• 3T5C