Chain-shortening enzymes

The retro-Claisen reaction occurs by initial nucleophilic addition of a cysteine -SH group on the enzyme to the keto group of the /3-ketoacyl CoA to yield an alkoxide ion intermediate. Cleavage of the C2-C3 bond then follows, with expulsion of an acetyl CoA enolate ion. Protonation of the enolate ion gives acetyl CoA, and the enzyme-bound acyl group undergoes nucleophilic acyl substitution by reaction with a molecule of coenzyme A. The chain-shortened acyl CoA that results then enters another round of tire /3-oxidation pathway for further degradation. 

The chain shortening pathway has not been characterized in detail at the enzymatic level in insects. It presumably is similar to the characterized pathway as it occurs in vertebrates. These enzymes are a partial P-oxidation pathway located in peroxisomes [29]. The key enzymes involved are an acyl-CoA oxidase (a multifunctional protein containing enoyl-CoA hydratase and 3-hy-droxyacyl-CoA dehydrogenase activities) and a 3-oxoacyl-CoA thiolase [30]. These enzymes act in concert to chain shorten acyl-CoAs by removing an acetyl group. A considerable amount of evidence in a number of moths has accumulated to indicate that limited chain shortening occurs in a variety of pheromone biosynthetic pathways. 

The above three examples illustrate how a species-specific pheromone blend is produced by the concerted action of desaturases, chain shortening enzymes, a reductase, and an acetyltransferase. The specificity inherent in certain enzymes in the pathway produces the final blend of pheromone components. 

In contrast to pheromones that involve single complex compounds, many moth species have been found to utilize a specific blend of relatively simple fatty acid-derived compounds. It appears that the evolution of a unique enzyme, A1 desaturase, used in combination with 2-carbon chain-shortening reactions (Figure 3) has allowed moth species to produce a variety of unsaturated acetates, aldehydes, and alcohols that can be combined in almost unlimited blends to impart species specificity. For example, biosynthetic precursors for the six-component pheromone blend of acetates for the cabbage looper moth (12) (Figure 2) can be determined easily from the cascade of acyl intermediates produced by the A11-desaturase and chain-shortening reactions (Figure 3). 

For the biosynthesis of vanillin, several other enzymes are of interest. First of all, phenylalanine ammonia lyase (PAL) this enzyme converts phenylalanine into the cinnamic acid type of compounds, the first intermediates in the vanillin biosynthesis after the primary metabolism. PAL activity could be detected in green beans, but after scalding this activity is lost. The chain shortening enzyme (CSE), responsible for the conversion of a C Cs compound into a C6Ci compound, was found to be localised in the cytosol of cells of the placental tri-chomes in the green beans [23]. 

The long-chain FAO disorders of CACT deficiency and CPT-II deficiency can not be differentiated because both cause accumulation of the same long-chain acylcarnitine species, which is explained by the fact that neither enzyme is involved in the chain-shortening action of FAO (Table 3.2.1 Fig. 3.2.6e and f). Isolated long-chain... 

Z11,E14-16 CoA, which is then chain shortened to Z9,E12-14 CoA, or alternatively Z1 l-16 CoA is chain shortened to Z9-14 CoA and then a A12 desaturase produces Z9,E12-14 CoA. Both pathways are possibilities based on the previous identification of a A14 desaturase in the Asian com borer (Zhao et al., 1990) and a A12 desaturase in some other insects (de Renobales et al., 1987). To differentiate between the two possible pathways, D9-Zll-16 acid (Z 11-[13,13,14,14,15, 15,16,16,16-2H9]hexadecenoic acid and D2-Z9-14 acid (Z9-[13,14-2H2] tetradecenoic acid) were applied to the glands. The results indicate that D9-Z11-16 acid was chain shortened to Z9-14 acid but was not converted to Z9,E12-14 CoA. This indicates that chain shortening occurs and incorporation of D2-Z9-14 acid into Z9,E12-14 CoA indicates that a A12 desaturase is present. The D9-Zll-16 acid was not incorporated into Z9,E12-14 CoAbecause of an isotope effect. This labeled compound had nine deuteriums on the methyl end where the desaturase enzyme would act, preventing the introduction of a double bond. Combined together, the labeling studies with precursors and intermediates indicate... 

The above examples illustrate the use of deuterium labeling to help determine the most likely pathway for biosynthesis of pheromone components. The key components of these pathways are fatty acid biosynthesis, desaturation, chain shortening and specific enzymes to produce a functional group. 

Changes in reductase activity could result in altered pheromone amounts. The lightbrown apple moth, Epiphyas postvittana, exhibits a decline in pheromone titers with age that is not due to reduced PBAN levels (Foster and Roelofs, 1994). To determine what is responsible for the decline in titers, different deuterium-labeled precursors were applied to the glands of different aged females. Biosynthesis of the major pheromone component, Ell-14 OAc, occurs by chain shortening 16 acid to 14 acid followed by Ell desaturation, reduction, and acetylation (Foster and Roelofs, 1990). Application of deuterium-labeled intermediates indicated that the senescent decline in pheromone titers was due to the reduction in fatty acid reductase activity (Foster and Greenwood, 1997). It is unknown if a decline in reductase enzyme activity is also responsible for observed senescent decline in pheromone titers of other female moths (Foster and Greenwood, 1997). 

After chain shortening, the 14-carbon and 12-carbon intermediates are reduced to an alcohol and acetylated. The acetyltransferase enzyme is not specific and will accept a variety of substrates (Jurenka and Roelofs, 1989). Unfortunately, the reductase has not been characterized at the enzymatic level. However, intermediate fatty alcohols are not found in the gland, therefore tight coupling of the reductase and acetyltransferase probably occurs. From these observations, it can be inferred that the final ratio of pheromone components is produced by the specificity found within the All desaturase and chain-shortening enzymes. 

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