Ketoacyl-ACP reductase

Acyl carrier protein (ACP) Acetyl-CoA-ACP transacetylase (AT) j3-Ketoacyl-ACP synthase (KS) Malonyl-CoA-ACP transferase (MT) )3-Ketoacyl-ACP reductase (KR) j8-Hydroxyacyl-ACP dehydratase (HD) Enoyl-ACP reductase (ER)... 

Step (2) Reduction of the Carbonyl Group The acetoacetyl-ACP formed in the condensation step now undergoes reduction of the carbonyl group at C-3 to form d-j8-hydroxybutyryl-ACP. This reaction is catalyzed by /3-ketoacyl-ACP reductase (KR) and the electron donor is NADPH. Notice that the D-j3-hydroxybutyryl group does not have the same stereoisomeric form as the l-j8-hydroxyacyl intermediate in fatty acid oxidation (see Fig. 17-8). 

The Reduction Reactions. The object of the next three reactions (steps 4 to 6 in fig. 18.12a) is to reduce the 3-carbonyl group to a methylene group. The carbonyl is first reduced to a hydroxyl by 3-ketoacyl-ACP reductase. Next, the hydroxyl is removed by a dehydration reaction catalyzed by 3-hydroxyacyl-ACP dehydrase with the formation of a trans double bond. This double bond is reduced by NADPH catalyzed by 2,3-trans-enoyl-ACP reductase. Chemically, these reactions are nearly the same as the reverse of three steps in the j6-oxidation pathway except that the hydroxyl group is in the D-configuration for fatty acid synthesis and in the L-configuration for /3 oxidation (compare figs. 18.4a and 18.12a). Also remember that different cofactors, enzymes and cellular compartments are used in the reactions of fatty acid biosynthesis and degradation. 

The active site cysteine of KS is represented in the figure by Cys-SH. The /3-ketobutyryl-ACP is reduced to the /3-hydroxy derivative by /3-ketoacyl-ACP reductase (KR, step 4 in fig. 18.12a), dehydrated to enoyl-ACP by /3-hydroxylacyl-ACP dehydrase (DFI, step 5 in fig. 

Fig. 5. Predicted domain organization and biosynthetic intermediates of the erythromycin synthase. Each circle represents an enzymatic domain as follows ACP, acyl carrier protein AT, acyl-transferase DH, dehydratase ER, P-ketoacyl-ACP enoyl reductase KR, [3-ketoacyl-ACP reductase KS, p-ketoacyl-ACP synthase TE, thioesterase. Zero indicates dysfunctional domain.

It has also been demonstrated that 3-ketoacyl-ACP reductase (FabG) can accept not only acyl-ACP but also acyl-CoA as a substrate and is capable of supplying mcl-(R)-3HA-CoA from fatty acid p-oxidation in Escherichia co/i. FabG, which is known as a homologue of PhaB, has been reported to serve as a monomer supplier for PHA biosynthesis in recombinant E. colt. 3-Ketoacyl-AGP synthase III (FabH) is also a constituent of fatty... 

Production of MCE PHAs was first investigated in recombinant E. coli by Langenbach et al (1997). A number of strategies have been developed to improve MCL PHA productivity by providing PHA precursors from the fatty acid P-oxidation pathway (Fig. 3 Park et al. 2004). The P-oxidation pathway has been engineered by the overexpression of enoyl-CoA hydratase (Fiedler et al. 2002 Fukui and Doi 1998) or 3-ketoacyl-ACP reductase (Park et al. 2002 Ren et al. 2000 ... 

Taguchi K, Aoyagi Y, Matsusaki H, Fukui T, Doi Y (2(X)3) Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HBlOl strain. FEMS Microbiol Lett 176 183-190 Tajima K, Igari T, Nishimura D, Nakamura M, Satoh Y, Munekata M (2003) Isolation and characterization of Bacillus sp. INT005 accumulating polyhydroxyalkanoate (PHA) from gas field soil. J Biosci Bioeng 95 77-81... 

The acetoacetate ester formed in the initial condensation step is reduced by 3-ketoacyl-ACP reductase in the presence... 

Fatty acid synthesis in bacteria and plants is a multistep process. One transformation in the process involves the reduction of the ketone unit in acetoacetyl AGP (94) with the enzyme [3-ketoacyl-ACP reductase and NADPH (91) to give (3-hydroxybutaryl-ACP (96) and NADP" (92). AGP is the acyl carrier protein, and it is bound to the acetoxy unit via a phosphopantetheine group (marked in cyan in 94). 

Although it was known that the intermediates of the yS-oxidation cycle are chaimelled towards PHA biosynthesis, only recently the precursor sources were identified. In A. caviae, the y3-oxidation intermediate, trans-2-tnoy -CoA is converted to (R)-3-hydroxyacyl-CoA via (R)-specific hydration catalysed by an (R)-specific enoyl-CoA hydratase [125, 126]. Subsequently, Tsuge and co-workers [127] reported the identification of similar enoyl-CoA hydratases in Pseudomonas aeruginosa. In the latter case, two different enoyl-CoA hydratases with different substrate specificities channelled both SCL and MCL enoyl-CoA towards PHA biosynthesis. In recombinant . coli it was further shown that 3-ketoacyl-CoA intermediates in the )8-oxidation cycle can also be channelled towards PHA biosynthesis by a nicotinamide adenine dinucleotide phosphate dependent (NADPH-dependent) 3-ketoacyl-ACP reductase [128]. A similar pathway was also identified in P. aeruginosa [129]. In addition, it was also reported that the acetoacetyl-CoA reductase (PhaB) of R. eutropha can also carry out the conversion of 3-ketoacyl-CoA intermediates in Pathway II to the corresponding (R)-3-hydroxyacyl- CoA in E. coli [130]. The results clearly indicate that several channelling pathways are available to supply substrates from the y3-oxidation cycle to the PHA synthase. This explains why it was not possible to obtain mutants that completely lack PHA accumulation ability, unless the mutation occurred in the PHA synthase gene [131]. 

In recent years, the molecular organization of the plant fatty acid synthetases has been examined in extracts from various plant tissues, and in all cases was found to be nonassociated and similar to the prokaryotic type of coli (1,2,3). Many of the individual component enzymes have been partially purified Including malonyl-CoAtacyl carrier protein (ACP) trans-acylase (4), 3-ketoacyl-ACP reductase (5,6), E-hydroxylacy1-ACP dehydrase (6), enoyl ACP reductase (5,6) and acetyl-CoA ACP transacylase (ATA) (1). 

Figure 22.4 A simplified illustration of saturated fatty acid biosynthesis in microalgal chloroplast. ACCase, Acetyl-CoA carboxylase ACP, acyl carrier protein CoA, coenzyme A ENR, enoyl-ACP reductase HD, 3-hydroxyacyl-ACP dehydratase KAR, 3-ketoacyl-ACP reductase KAS, 3-ketoacyl-ACP synthase MAT, malonyl-CoA ACP transacylase.

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