Malonyl-ACP

The enzymes that catalyze formation of acetyl-ACP and malonyl-ACP and the subsequent reactions of fatty acid synthesis are organized quite differently in different organisms. We first discuss fatty acid biosynthesis in bacteria and plants, where the various reactions are catalyzed by separate, independent proteins. Then we discuss the animal version of fatty acid biosynthesis, which involves a single multienzyme complex called fatty acid synthase. 

The individual steps in the elongation of the fatty acid chain are quite similar in bacteria, fungi, plants, and animals. The ease of purification of the separate enzymes from bacteria and plants made it possible in the beginning to sort out each step in the pathway, and then by extension to see the pattern of biosynthesis in animals. The reactions are summarized in Figure 25.7. The elongation reactions begin with the formation of acetyl-ACP and malonyl-ACP, which... 

The net result of this biosynthetic cycle is the synthesis of a four-carbon unit, a butyryl group, from two smaller building blocks. In the next cycle of the process, this butyryl-ACP condenses with another malonyl-ACP to make a... 

Claiscn-like condensation of malonyl ACP with acetyl synthase occurs, followed by decarboxylation to yield acetoacetyl ACP, a -keto thioester. 

Following the formation of malonyl CoA, another nucleophilic acyl substitution reaction occurs in step 4 to form the more reactive malonyl ACP, thereby binding the malonyl group to an ACP arm of the multienzyme synthase. At this point, both acetyl and malonyl groups are bound to the enzyme, and the stage is set for their condensation. 

Step 5 of Figure 29.5 Condensation The key carbon-carbon bond-forming reaction that builds the fatty-acid chain occurs in step 5. This step is simply a Claisen condensation between acetyl synthase as the electrophilic acceptor and malonyl ACP as the nucleophilic donor. The mechanism of the condensation is thought to involve decarboxylation of malonyl ACP to give an enolate ion, followed by immediate addition of the enolate ion to the carbonyl group of acetyl... 

Thiolactomycin (16) is another natural product that reversibly inhibits E. coli FabF, FabB, and FabH with respective ICso s of 6, 25 and 110 (iM. Unlike cerulenin, it binds the malonyl-ACP site of the enzyme [27]. Despite modest double-digit MICs on . coli, S. aureus, Serratia marces-cens, and Mycobacterium tuberculosis, 16 has generated quite some interest due to its good in vivo protection against an oral or intramuscular S. marcescens urinary tract infection model where it displayed rapid tissue distribution [28]. Despite several medicinal chemistry efforts, thiolactomycin has proven difficult to optimize due to some strict functional group requirements for its SAR [29]. 

Malonyl-ACP, formed from acetyl-CoA (shuttled out of mitochondria) and C02, condenses with an acetyl bound to the Cys—SH to yield acetoacetyl-ACP, with release of C02. This is followed by reduction to the D-/3-hydroxy derivative, dehydration to the trans-A2-unsaturated acyl-ACP, and reduction to butyryl-ACP. NADPH is the electron donor... 

Six more molecules of malonyl-ACP react successively at the carboxyl end of the growing fatty acid chain to form palmitoyl-ACP—the end product of the fatty acid synthase reaction. Free palmitate is released by hydrolysis. 

Both bacteria and plants have separate enzymes that catalyze the individual steps in the biosynthetic sequence (Fig. 17-12). The fatty acyl group grows while attached to the small acyl carrier protein (ACP).54 58 Control of the process is provided, in part, by the existence of isoenzyme forms. For example, in E. coli there are three different P-oxoacyl-ACP synthases. They carry out the transfer of any acyl primer from ACP to the enzyme, decarboxylate malonyl-ACP, and carry out the Claisen condensation (steps b, e, and/in Eq. 17-12)58a e One of the isoenzymes is specialized for the initial elongation of acetyl-ACP and also provides feedback regulation.58c The other two function specifically in synthesis of unsaturated fatty acids. 

The Condensation Reaction. In the condensation reaction the acetyl group is initially transferred from ACP on to a SH group of 3-ketoacyl-ACP synthase. This acetyl moiety then reacts with malonyl-ACP (step 3 in fig. 18.12 ) so that the acetyl component becomes the methyl terminal two carbon unit of the acetoacetyl-ACP. The release of C02 in this condensation reaction provides the extra thermodynamic push to make the reaction highly favorable. 

Figure 8 Decarboxylation of malonyl-ACP by the CLF. Decarboxylation of malonyl-ACP by the CLF results in acetyl-ACP, an enzyme-bound starter unit. Initiation of chain extension can then occur by transfer of the acetate starter unit to the KS domain.

P Zhou, G Florova, KA Reynolds. Polyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis. Chem Biol 6 577-584, 1999. 

The pathway The first committed step in fatty acid biosynthesis is the carboxylation of acetyl CoA to form malonyl CoA which is catalyzed by the biotin-containing enzyme acetyl CoA carboxylase. Acetyl CoA and malonyl CoA are then converted into their ACP derivatives. The elongation cycle in fatty acid synthesis involves four reactions condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP releasing free ACP and C02, then reduction by NADPH to form D-3-hydroxybutyryl-ACP, followed by dehydration to crotonyl-ACP, and finally reduction by NADPH to form butyryl-ACP. Further rounds of elongation add more two-carbon units from malonyl-ACP on to the growing hydrocarbon chain, until the C16 palmitate is formed. Further elongation of fatty acids takes place on the cytosolic surface of the smooth endoplasmic reticulum (SER). 

CoA to form malonyl CoA using C02 in the form of bicarbonate HC03 (Fig. 2). This reaction is catalyzed by the enzyme acetyl CoA carboxylase which has biotin as a prosthetic group, a common feature in C02-binding enzymes. One molecule of ATP is hydrolyzed in the reaction, which is irreversible. The elongation steps of fatty acid synthesis all involve intermediates linked to the terminal sulfhydryl group of the phosphopantetheine reactive unit in ACP phosphopantetheine is also the reactive unit in CoA. Therefore, the next steps are the formation of acetyl-ACP and malonyl-ACP by the enzymes acetyl transacylase and malonyl transacylase, respectively (Fig. 2). (For the synthesis of fatty acids with an odd number of carbon atoms the three-carbon propionyl-ACP is the starting point instead of malonyl-ACP.)... 

Condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP, releasing free ACP and C02 (catalyzed by acyl-malonyl-ACP condensing enzyme). 

This first round of elongation produces the four-carbon butyryl-ACP. The cycle now repeats with malonyl-ACP adding two-carbon units in each cycle to the lengthening acyl-ACP chain. This continues until the 16-carbon palmitoyl-ACP is formed. This molecule is not accepted by the acyl-malonyl-ACP condensing enzyme, and so cannot be elongated further by this process. Instead it is hydrolyzed by a thioesterase to give palmitate and ACP. 

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