CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
1.8 Overview of acetyl-CoA carboxylase
Acetyl-CoA carboxylase (ACC) [EC 6.4.1.2] is an enzyme that catalyses the carboxylation of acetyl-CoA to form malonyl-CoA in the rate limiting step of de novo fatty acid biosynthesis. This reaction is driven by ATP hydrolysis (Abu-Elheiga et al., 2000; Abu-Elheiga et al., 1995; Al-Feel et al., 1992; Brownsey et al., 2006; Lu et al., 2005; Wakil et al., 1983). In addition to being involved in fatty acid biosynthesis, ACC may be involved in the synthesis of polyketides and possibly in yet unknown biosynthetic pathways leading to the production of unknown compounds (Al-Feel et al., 1992; Hopwood and Sherman, 1990; Sasaki et al., 1993). The product, malonyl- CoA, formed by ACC, becomes an important substrate for fatty acid synthesis in the liver and lactating mammary glands of animals. Malony-CoA is a substrate for the chain elongation system of enzymes in the endoplasmic reticulum and in many tissues with fatty acid chains greater than 20 subunits. Malonyl-CoA is involved in numerous cellular functions such as the synthesis of eicosanoid, glycolipids, sphingolopids and glycerolipids. Malonyl-CoA also plays an important function in the regulation of fatty acid oxidation by inhibiting palmitoyl-CoA transferase I (Brownsey et al., 2006). Therefore the availability of malonyl-CoA can alter the rate of fatty acid biosynthesis, β-oxidation and ketogenesis (McGarry, 2002). By and large, the role played by ACC in the production of malonyl-CoA emphasises the importance of ACC in carbon and energy metabolism in organisms expressing this enzyme.
Figure 1.4 illustrates the rate limiting step in fatty acid biosynthesis catalysed by ACC. The enzyme transfers a carboxylate ion (COO¯ ) to acetyl-CoA (Boone et al., 2000; Brownsey et al., 2006). The first step is the ATP-dependent phosphorylation of bicarbonate to carbamoyl phosphate. This step is catalysed by the carbamoyl phosphate synthatase sub-domain of the biotin carboxlase domain (Boone et al., 2000; Brownsey et al., 2006). In the second step, the phosphate- activated carboxylate ion is transferred to the biotin moiety in the biotinoyl domain to form carboxybiotinyl ACC. The carbondioxide is subsequently detached from the biotin molecule and transferred to acetyl-CoA in the final step. This step is catalysed by the carboxyl transferase domain (Boone et al., 2000; Brownsey et al., 2006).
In mammals, there are two isoforms of ACC namely ACC1 (ACC α) and ACC2 (ACC β), as illustrated in Figure 1.5. These two isoenzymes vary in their amino acid sequence suggesting
possible variation in function between these two isozymes. ACC1 is expressed in the lipogenic tissues as a ~ 265 kD monomeric protein, where it is involved in the rate limiting step of fatty acid biosynthesis. ACC2 is a 270 – 280 kD monomeric protein expressed in the skeletal and heart muscle where is it believed to play an important role in the regulation of fatty acid oxidation (Brownsey et al., 2006; Ha et al., 1996; Lu et al., 2005).
HO O O
ADP O P O O
O
HO O O
P O O
O
ACC N
S HN NH
O
O
N S
HN N
O
O
O O
ACC
CoA S H
H O
CoA S
O O O Bicarbonate
ADP ATP
Carbamoyl-phosphate
Biotinyl ACC
Carboxybiotinyl ACC Acetyl-CoA
Malonyl-CoA
Pi 1
2
3 _
_
_
_ _
_
_
Figure 1.4 Formation of malonyl-CoA from acetyl-CoA and bicarbonate. Step 1 is the phosphorylation of the carboxylate ion to form carbamoylphosphate; Step 2 is the transfer of carbamoylphosphate to the biotin molecule on the biotinoyl domain to form carboxybiotinyl ACC; Step 3 is the transfer of carboxylate ion to acetyl-CoA to form malonyl-CoA. Steps 1 and 2 are catalysed by the biotin carboxylase domain (or subunit) and Step 3 is catalysed by the carboxyl transferase domain.
The complex structure of ACC may suggest the complex nature of its catalytic and regulatory properties. ACC found in higher animals, protozoa (Toxoplasma gondii), plants and yeasts have three protein domains. The biotin carboxylase domain is situated at the N-terminal region of the enzyme. The biotin/lipoyl binding domain (the biotin carboxyl carrier protein or biotinoyl domain) is situated closer to the biotin carboxylase domain at the N-terminal region of the enzyme. The carboxyltransferase domain is situated at the C-terminal region. In some organisms including mammals, the carboxyltransferase domain is further subdivided into an N-terminal transferase and a C-terminal transferase domain (Brownsey et al., 2006; Cronan and Waldrop, 2002; Ha et al., 1996; Lu et al., 2005). ACC exists in the form of a dimer or polymer of up to 4 000 Å unit (~ 400 nm in length). In vitro studies have shown that the dimeric form of ACC shows low catalytic activity while the polymeric form is more active (Brownsey et al., 2006).
Figure 1.5 Domain organisation of the multi-enzyme domain acetyl-CoA carboxylase. (A) Acetyl- CoA carboxylase 1 (ACC 1) (B) Acetyl-CoA carboxylase 2 (ACC 2) with the mitochondrial signal sequence (filled box) at the N-terminal (Hardie and Pan, 2002).
Biotin
C N
Biotin carboxylase Biotin carboxyl Carboxyl transferase carrier protein
A
Biotin
N C
Biotin carboxylase Biotin carboxyl Carboxyl transferase carrier protein
B
ACC2 has a predicted molecular weight of 290 kD; larger than ACC1. ACC2 was first isolated from rat heart (Thampy, 1989; Thampy and Wakil, 1988a; Thampy and Wakil, 1988b). Sequence analysis of ACC2 showed that it has an N-terminal sequence extension. This sequence (not found in ACC1), which contains a mitochondrial targeting motif, differentiates ACC1 from ACC2.
ACC is made up of an ATP binding motif, a protein kinase recognition site and other numerous catabolite binding sites that are involved in the regulation of the enzyme. Three dimensional structural studies have been reported for the biotin carboxyl carrier protein of E. coli and Saccharomyces cerevisiae (Roberts et al., 1999; Waldrop et al., 1994) as well as the carboxyl transferase domain of Saccharomyces cerevisiae (Zhang et al., 2003). To date, there is no resolved 3D structure for mammalian, plant and protozoan ACC domains. The important nature of this enzyme in various organisms as a “manager” of carbon and energy metabolism makes it attractive to resolve its three dimensional structure.
Some plants express both the multi-enzyme-domain type ACC as well as the multi-enzyme- subunit type ACC complex. The multi-enzyme-subunit type ACC complex form is localised in the plastid of plant cells where de novo fatty acid biosynthesis takes place, and the multi-enzyme- domain ACC is localised in the plant cell cytosol. De novo fatty acid biosynthesis found in plants is very similar to the pathway of yeast, and higher animals (Konishi et al., 1996; Sasaki et al., 1993; Zuther et al., 1999). The plant plastid multi-enzyme-subunit ACC complex (similar to those found in prokaryotes) dissociates into four protein subunits namely: the biotin carboxyl carrier protein (biotinoyl protein), biotin carboxylase, α-carboxyltransferase and β- carboxyltransferase (Figure 1.6). The plastid ACC is involved only in fatty acid biosynthesis, while the cytosolic ACC is involved in flavonoid, anthocyanin, malonated amino acids, very long chain fatty acid biosynthesis and other natural products found in plants (Sasaki and Nagano, 2004; Thelen et al., 2001).
Grass and some monocotyledonous plants lack the gene that codes for the multi-enzyme-domain ACC subunits complex, but encodes the multi-enzyme-domain ACC that is targeted to both the plastid and the cytosol. The lack of the multi-enzyme-subunit ACC complex in grasses confirms the sensitivity shown towards herbicides by these plants. Herbicides such as fenoxaprop, diclofop and sethoxydim (Figure 1.8) inhibit the activity of the multi-enzyme-domain ACC, while the
multi-enzyme-subunit ACC complex is insensitive to herbicides (Davis et al., 2000). There is no defined molecular weight of plant ACC. But studies have shown that the plant single multi- enzyme-domain ACC is in the form of a dimer ranging from about 210 kD to 250 kD. The observed size in pea ACC is about 650 – 700 kD (Sasaki and Nagano, 2004). The usual flow of carbon from photosynthesis to primary and secondary metabolites in plants is controlled by ACC.
Therefore in plants, ACC and malonyl-CoA (product of ACC) are vital for the regulation of metabolite flux through the fatty acid biosynthetic and degradation pathways (Jelenska et al., 2001; Jelenska et al., 2002).
Figure 1.6 The multi-enzyme acetyl-CoA carboxylase complex (Sasaki and Nagano, 2004).