Introduction

Mycobacterium tuberculosis very-long-chain fatty acyl-CoA synthetase, also known as FadD13, is unique within its class of FadD proteins in regards to its ability to house lipid substrates longer than itself. These lipid substrates are very-long-chain fatty acids between lengths C22 –C26, which is up to the maximum length tested. ^[1] The significance of theses very-long-chain fatty acids lies in their importance to mycolic acid synthesis by Mycobacterium tuberculosis (M. tb). Mycolic acids compose part of the cell wall of (M. tb). FadD13, an activator of mycolic acids, has been identified as key component in the virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis, and has emerged as possible target for novel therapeutic agents.^[2] The FadD13 enzyme is the last gene of the mymA operon. ^[3]

There are four main groups of FadD enzymes categorized on their ability to accommodate different length substrates: short (C2-C4), medium (C4-C12), long (C12-C22), and very long (C22-C26).^[1] Most FadD class proteins exist as integral membrane proteins, involved in both the activation of fatty acids and other hydrophobic substrates in addition to the transport of these lipids into the cell. However, substrates longer than the enzyme itself, like these very-long-chain fatty acids, pose and interesting structural dilemma to the enzyme. In order for FadD13 to accommodate these very long substrates, it differs structurally in that it exists as a peripheral protein on the inside of the cell membrane. This feature is key to the mechanistic basis for FadD13’s activation and transport of fatty acids of length C24-C26 through the two step addition of Coenzyme A(Figure 1).^[1]

Mechanism

Figure 1: Mechanism for the activation of fatty acids (C24-C26) by FadD13. The N terminal domain (pink) is embedded in the membrane with the arginine rich lid-loop (dark blue), while the flexile linker (black) connects the C terminal domain (green) to the rest of the enzyme. Activation requires the binding of ATP (blue) which induces structural changes that promote the binding of the fatty acid chain. Formation of an acyl-adenylate intermediate induces a 140° rotation of the C terminal domain and the binding of CoA (orange).

Figure 2: Representation of the two-step reaction catalyzed by FadD13

General mechanism for the activation of fatty acids

FadD13 represents the first Fatty Acyl-CoA Synthetase of its kind to display biphasic kinetics.^[3] FadD13 first activates the fatty acid through a reaction with ATP to form an acyl adenylate intermediate and subsequently releases a pyrophosphate. Following a conformational change of the enzyme upon the binding of ATP, coenzyme A is able to bind to its active site and react with the acyl adenylate intermediate forming the acyl CoA product (Figure 2). These activated fatty acyl-CoA thioesters have then been demonstrated to be important for the synthesis of triacyglycerols and phospholipids in the membrane of Mycobacterium tuberculosis. ^[3]

Structural basis for housing lipid substrates longer than the enzyme

The ability for FadD13 to transport and activate fatty acids of the maximum tested length C26, lies in it being a peripheral membrane protein. FadD13's attachment to the membrane via electrostatic interactions in the N-terminal domain is coupled with the presence of a hydrophobic tunnel located centrally in this same domain. This method of attachment, with the alignment of the hydrophobic tunnel to the membrane, allows the extension of these very-long-chain fatty acids to enter FadD13 from the membrane (Figure 1). Of importance to the passage of these fatty acid substrates into FadD13 resides in the presence of an arginine rich lid-loop, located at the top of the hydrophobic tunnel and embedded in the membrane. Once the lid-loop is opened, fatty acids may be pulled from the membrane into a hydrophobic tunnel, which is the main structural component by which fatty acids are capable of transportation from the membrane into the enzyme (Figure 1).

Structure

FadD13 is composed of 503 amino acid residues divided into three main regions: The (residues 1-395) and (residues 402-503) which are connected via a flexible represented in dark blue (residues 396-401).^[1] Each region plays an important role in the activation of fatty acids. The large N-terminal domain houses the main key structural features involved in fatty acid activation, but ultimately it is the flexible linker that allows movement of the C-terminal domain to from the fully functioning active site of FadD13 (Figure 1).

Electrostatics

Figure 3: Pmyol depiction of electrostatic interactions of FadD13.

The electrostatics of FadD13 as seen in (Figure 3) illustrate the hydrophobic and positively charged regions that compose this protein. Experimental results revealed that the peripheral FadD13 is attached to the membrane via electrostatic and hydrophobic regions located on the top portion of the N-terminal region (Figure 3).^[1] Of key importance in this N-terminal domain region attached to the membrane is an area of notable arginine rich residues, known as the arginine rich lid-loop.

Arginine Rich Lid-loop

The functions to block entry of fatty acids into the hydrophobic tunnel of FadD13.This area on the top portion of the enzyme is also crucial in the association with the membrane as the positively charged arginine residues are attracted to the negative charge on the phospholipid heads.^[1]

Hydrophobic Tunnel

The hydrophobic tunnel of FadD13 is essential to the transport and accommodation of very long fatty acids from the membrane into the cell. This tunnel runs through the middle of FadD13 from the arginine rich lid loop to the ATP binding site and is situated between the and alpha helices α8-α9 and parallel beta sheet β9- β14 (Figure 3).^[1] Negatively charged residues at the active site of FadD13 are the driving factor in the attraction of the fatty acid from the membrane through the hydrophobic tunnel of the enzyme.

Figure 4: Pmyol depiction of hydrophobic tunnel.

Active Site

The FadD13 active site is composed of positively charged regions which account for the attraction and binding of hydrophobic substrates to this region (Figure 3). The active site on FadD13 is composed of two conserved regions, one of which serves as the binding site for ATP and the other for CoA. The adenine of ATP is bound to a group of that is structurally identically to other acyl-CoA synthetases. ^[1]

Mutational studies showed that highly conserved residue in the C-terminal region, , resulted in a 95% loss of function of FadD13 and is thought to be involved in the orientation of the substrates to form the adenylate intermediate.^[3] Additionally, Serine 404 was hypothesized to be involved in the binding of Coenzyme A which may only occur once this region incurs a 140 degree rotational change after the initial binding of ATP.^[1][3]

Disease

Mycobacterium tuberculosis (M.tb) is the causative agent involved in the disease tuberculosis. Tuberculosis is a growing global health concern that has been intensified due to the increase in HIV infections along with the increase in multi-drug resistance strains of (M. tb) ^[4]. Most of the drug resistance has evolved due to the intensive nature of the treatment for tuberculosis, which often goes incomplete thus resulting in drug resistant strains; therefore, the importance in identifying characteristics and residues to be exploited for new drug targets is pivotal ^[4].

The cell wall of (M. tb) is known to be composed and synthesized from a distinct variety of lipids, most notably mycolic acids, which are known to play a crucial role in the pathogenesis of (M. tb) ^[4] The mycolic acid biosynthetic pathway has been proposed to involve five distinct stages, the first of which is the synthesis of C20 to C26 straight-chain saturated fatty acids activated by FadD13. ^[5] For this reason, current research has focused on inhibitors to disrupt the initiation of this biosynthetic pathway of mycolic acids in (M. tb).

Current Treatment

Drugs developed thus far that have been shown to inhibit mycolic acid biosynthesis are: isoniazid, ethionamide, isoxyl, thiolactomycin, and triclosan. ^[5] Additionally, pyrazinamide was shown to inhibit fatty acid synthase type I which is involved in providing a precursor necessary for fatty acid elongation to long-chain mycolic acids. ^[5] Treatment for active cases of tuberculosis include the simultaneous therapeutic use of two or more frontline drugs: isoniazid, ethambutol, rifampicin and pyrazinamide. ^[4]

Future Research

While currently there are no specific drug targets for FadD13, a better understanding of key residues involved in the activation of very-long-chain fatty acids is a promising start to developing new drug targets for (M. tb). Recent studies have shown the mymA operon, which is involved in the maintenance of (M. tb) cell wall architecture, and which codes for the enzyme FadD13, is up-regulated under acidic conditions. ^[4] Functional loss of the mymA operon resulted in increased drug sensitivity and death of the pathogen; therefore any drugs that can can target this operon will be effective at fighting tuberculosis. ^[4]