Condensing enzyme plant

Millar A. A. and Kunst L. (1997) Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J. 12, 121-131. 

Dehesh, K., Edwards, R, Fillatti, J., Slabaugh, M. and Byrne J. 1998. KAS IV A 3-ketoacyl-ACP synthase from Cuphea sp. is a medium chain specific condensing enzyme. Plant J. 15 383-390. 

Chalcone synthases (CHS) and stilbene synthase (STS) are closely related plant PKSs that catalyze the stepwise condensation between acyl CoA esters in the biosynthesis of flavonoids, stilbenes, and other related plant aromatic polyke-tides [70,72,75]. Sequences for munerous CHS as well as a few STS have been determined from various plants [72-76, 122-131]. They all possess a highly conserved cysteine (Cys) residue that is essential for PKS activity, although the sequences in this Cys motif have no apparent similarity to that of KS of bacterial and fungal PKSs [ 132]. The plant PKSs are essentially condensing enzymes,lack the ACP domain, and use the acyl CoA esters directly as substrate for the condensing reactions [72,75]. 

The biochemistry and enzymology of CHS and its closely related plant PKSs have been extensively studied [72, 75]. The native enzymes have been isolated from various plants [70,71,73,74,124,125,127,128], and the recombinant proteins have been produced in E. coli and purified [71,76,124,125,132-135,139]. The active site for the condensation reaction has been mapped to a single Cys residue [132]. The dimeric nature of CHS and STS has been established with each homodimer to constitute two active sites [76,127,133]. The subtle differences among the CHS and STS proteins have been compared, which seems to have a profoimd effect on substrate specificity and regiospedfidty for the condensation and cyclization reactions, respectively [74, 127, 128, 133, 134]. The interactions between CHS and other proteins, such as KR [133,136-139] and methylmalonyl CoA-specific condensing enzyme [76], have been examined to account for the biosynthesis of deoxychalcones and methylchalcones. 

Figure 1. Confirmation of stable integration of jojoba elongase condensing enzyme gene in the alfalfa genome using PCR. Genomic DNAs of both transformed and nontransformed plants were used to amplify an intervening region (indicated by arrow).

Figure 2. RNA blot analysis of jojoba elongase condensing enzyme gene expression in transgenic alfalfa. Total RNAs ( 15 gg) were separated on a formaldehyde gel, transferred onto a nylon membrane and hybridized with P-labeled jojoba condensing enzyme cDNA. RNA from vector transformed and nontransformed tissue culture-regenerated plants were used as control.

Walsh, M.C., Klopfenstein, W.E. and Harwood, J.L. (1990) The short chain condensing enzyme has a widespread occurrence in the fatty acid synthetases from higher plants. Phytochemistry 29, 3797-3799. 

Reactions 35 and 36 have been shown to be catalyzed by different enzyme fractions and the intermediate has been isolated and partially purified. Earlier reports had indicated that reaction 36 was a hydrolytic step resulting in the formation of arginine plus malic acid. However, recent studies indicate that the formation of malic acid was the result of the presence of fumarase in the enzyme preparation. Of considerable interest is the report that a compound similar to, if not identical with, the end product of reaction 35 is enzymatically formed from arginine plus fumaric acid by extracts of plant and animal tissues, and microorganisms. In this connection it has been reported that one of the components of the condensing enzyme system (reaction 35) is present in yeast extracts as well as in liver preparations. Although ATP is required for synthesis of the intermediate from citrulline plus aspartic acid, it is not needed for the synthesis from arginine plus fumaric acid. 

Textor S, Bartram S, Kroymann J, Falk KL, Hick A, Pickett JA, Jonathan Gershenzon J (2004) Biosynthesis ofmethionine-derived glucosinolates in Arabidopsis thaliana recombinant expression and characterization of mefliylthioalkylmalate synthase, the condensing enzyme of the chain-elongation cycle. Planta 218 1026-1035 Thaler J S (1999) Jasmonate-inducible plant defences cause increased parasitism of herbivores. Nature 399 686-688 Thackaray DJ, Wratten SD, Edwards PJ, Niemeyer HM (1990) Resistance to the aphids Sitobion avenae and Rhopalosiphum padi in Gramineae in relation to hydroxamic acid levels. Ann Appl Biol 116 573-582... 

Three condensing enzymes have been characterized in Escherichia coli hitherto. KAS I encoded by fabB is highly sensitive to cerulenin and is unique in its ability to carry out the first condensation of the unsaturated pathway. KAS II determined by fabF has an intermediate sensitivity to cerulenin in vivo, and is singular in accomplishing the final condensation of the unsaturated pathway. KAS III coded for by fabH appears to be very closely related to plant KAS III. 

Figure 3 The KAS complementation assay uses crude E. coli extracts. Condensing enzymes in varying stages of purification and in circa the same concentration as those inactivated by cerulenin treatment are added to the extract. The ability of the extract to incorporate radio label from " C-malonyl-CoA ( ) into elongated acyl chains is assayed. Acyl products are shown at bottom. Left to right before cerulenin treatment all four KASes (I-IV) are active after cerulenin treatment only KAS HI is active after cerulenin treatment KAS IV is added after cerulenin treatment both KAS I and IV are added. In the latter case if a plant protein fraction containing KAS II had been added simultaneously Cis chains would also be synthesized.

In Figure 4A the condensing enzymes of E. coli are placed in the fatty acid biosynthetic pathway according to our present concept of their preferred primer substrates [29]. A potential role for the newest family member (KAS IV) is in synthesis of Cg-acyl chains which are precursors of lipoic acid and the lipoamide cofactor [30]. Interesting questions can be posed such as when in evolution did KAS IV appear in bacteria and does a KAS IV occur in plants Older observations in the literature can be interpreted in support of the latter notion [31], as can the endosymbiont theory for the origin of plastids and the many similarities in fatty acid biosynthetic systems of plastids and E. coli. 

Slabaugh M, Leonard J, Tai H, Jaworski JG, Knapp S. Condensing enzymes and thioesterases expressed in immature embryos of Cuphea wrightii. Plant Lipid Symposium 1993 July 29-31 Minneapolis, MN Abst. A9 (Abstract)... 

Tai H, Jaworski JG. 3-ketoacyl-acyl carrier protein synthase III from spinach (Spinacea oleraced) is riot similar to other condensing enzymes of fatty acid synthase. Plant Physiol 1993 103 1361-1367. 

INHIBITION OF FATTY ACID CONDENSING ENZYMES IN PLANTS... 

Fatty acid synthesis in plants is carried out by a type I dissociable fatty acid synthase (FAS). There are three p-ketoacyl-ACP synthases (KAS) associated with FAS in plants. The short-chain condensing enzyme (KAS III) catalyses the initial condensation of acetyl-CoA with malonyl-ACP [1,2] to form acetoacetyl-ACP (4C) and may catalyse further rounds of condensation in vivo [3]. Further rounds of condensation are carried out by KAS I which catalyses the condensation of malonyl-ACP with intermediate length acyl-ACPs from acetoacetyl-ACP to myristoyl-ACP (14C) to give palmitoyl-ACP (16C). KAS II elongates palmItoyl-ACP (16C) to stearoyl-ACP (18C) [4J. 

There are some known inhibitors of fatty acid synthesis. Cerulenin inhibits KAS I completely and irreversibly at 20 j.M [5] and inhibits KAS II but at higher concentrations. Cerulenin also inhibits the condensing enzyme function of multifunctional protein type I fatty acid synthases [6]. For all these enzymes the mechanism of action Is similar, involving the covalent binding of the inhibitor to the cysteine of the active site of the protein [7]. Arsenite, an inhibitor of enzymes containing vicinal thiol groups, inhibits KAS II but no other condensing enzyme. KAS III is insensitive to inhibition by either cerulenin or arsenite but its activity is inhibited by thiolactomycin (TLM). This inhibition has been seen in bacteria [8] and plants [9] but is rather variable as reported in the literature [10-14]. 

In higher plants fatty acid biosynthesis takes place primarily in plastids. Three condensing enzymes (/3-ketoacyl-ACP synthases, KASes), namely KAS I, KAS II and KAS III, have been described. They are different with respect to the preferred length of fatty acyl substrates as well as in their sensitivity to the inhibitor cerulenin. 

Wissenbach M, Siggaard-Andersen M, Kauppinen S, Wettstein-Knowles P von. Condensing enzymes of barley. In Cherif A, Miled-Daoud DB, Marzouk B, Smaoui A, Zarrouk M, editors. Metabolism, Structure and Utilization of Plant Lipids. 1992 393-396. 

Three condensing enzymes (KASes) have been characterized from the fatty acid synthases of plants and E. coli. Recently we have found an E. coli KASIV with specificity for short chain acyl-ACPs and a high sensitivity to cerulenin. We are now investigating the possibility of a similar enzyme existing in plants. Here we describe both our basic KAS assay, as well as some modifications that expand the substrate specificity range over which the assay is useful. In this initial work we have also observed a possible compartmentalization of KASes which may further add to the complexity of plant FASes. 

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