Erythronolide synthase

Essential components Ketosynthase The active site of this enzyme has a very broad specificity, which allows for the synthesis of long chains of carbon atoms by joining, via a thioester linkage, small organic acids, such as acetic and malonic acid. The KS domain receives the growing polyketide chain from the upstream module and subsequently catalyzes formation of the C-C bond between this substrate and an ACP-bound extender unit that is selected by the AT domain. Acyltransferase Each AT domain has an α-carboxylated CoA thioester (i.e. methylmalonyl-CoA) This specificity prevents non-essential addition of enzymes within the module. The AT captures a nucleophilic β-carboxyacyl-CoA extender unit and transfers it to the phosphopantetheine arm of the ACP domain. Functions via catalyzing acyl transfer from methylmalonyl-CoA to the ACP domain within the same module via a covalent acyl-AT intermediate. The importance of the AT to the stringent incorporation of specific extender unit in the synthesis of polyketide building blocks makes it vital that the mechanism and structure of these domains be well-elucidated in order to develop efficient strategies for the regiospecific engineering of extender unit incorporation in polyketide biosynthesis. The ACP first accepts the extender unit from the AT, then collaborates with the KS domain in chain elongation, and finally anchors the newly elongated chain as it undergoes modification at the β-keto position. In order to carry out their function, the ACP domains require post-translational addition of a phosphopantetheine group to a conserved serine residue of the ACP. The terminal sulfhydryl group of the phosphopantetheine is the site of attachment of the growing polyketide chain. Thioesterase Located at the C-terminus site of the furthest downstream module. It is terminated in a thioesterase, which releases the mature polyketide (either as the free acid or a cyclized product), via lactonization. Note: As stated above, the first module of DEBS contains an additional acyltransferase and ACP for initiation of the reactions Non-essential components Additional components, may have any one or a combination of the following: Ketoreductase- Uses NADPH to stereospecifically reduce it to a hydroxyl group Dehydratase- Catalyzes the removal of the hydroxyl group to create a double bond from organic compounds in the form of water Enolreductase- Utilizes NADPH to reduce the double bond from the organic compound ==Comparison between fatty acid synthesis and polyketide synthesis==

Fatty acid synthesis in most prokaryotes occurs by a type II synthase made of many enzymes located in the cytoplasm that can be separated. However, some bacteria such as Mycobacterium smegmatis as well as mammals and yeast use a type I synthase which is a large multifunctional protein similar to the synthase used for polyketide synthesis. This Type I synthase includes discrete domains on which individual reactions are catalyzed. In both fatty acid synthesis and polyketide synthesis, the intermediates are covalently bound to ACP, or acyl carrier protein. However, in fatty acid synthesis the original molecules are Acyl-CoA or Malonyl-CoA but polyketide synthases can use multiple primers including acetyl-CoA, propionyl-CoA, isobutyryl-CoA, cyclohexanoyl-CoA, 3-amino-5-hydroxybenzoyl-CoA, or cinnamyl-CoA. In both fatty acid synthesis and polyketide synthesis these CoA carriers will be exchanged for ACP before they are incorporated into the growing molecule. During the elongation steps of fatty acid synthesis, ketosynthase, ketoreductase, dehydratase, and enoylreductase are all used in sequence to create a saturated fatty acid then postsynthetic modification can be done to create an unsaturated or cyclo fatty acid. However, in polyketide synthesis these enzymes can be used in different combinations to create segments of polyketide that are saturated, unsaturated, or have a hydroxyl or carbonyl functional group. There are also enzymes used in both fatty acid synthesis and polyketide synthesis that can make modifications to the molecule after it has been synthesized. As far as regulating the length of the molecule being synthesized, the specific mechanism by which fatty acid chain length remains unknown but it is expected that ACP-bound fatty acid chains of the correct length act as allosteric inhibitors of the fatty acid synthesis enzymes. In polyketide synthesis, the synthases are composed of modules in which the order of enzymatic reactions is defined by the structure of the protein complex. This means that once the molecule reaches the last reaction of the last module, the polyketide is released from the complex by a thioesterase enzyme. Therefore, regulation of fatty acid chain length is most likely due to allosteric regulation, and regulation of polyketide length is due to a specific enzyme within the polyketide synthase. ==Application==

Since the late 1980s and early 1990s research on polyketide synthases (PKS), a number of strategies for the genetic modification of such PKS have been developed and elucidated. Such changes in PKS are of particular interest to the pharmaceutical industry as new compounds with antibiotic or other antimicrobial effects are commonly synthesized after changes to the structure of the PKS have been made. Engineering the PKS complex is a much more practical method than synthesizing each product via chemical reactions in vitro due to the cost of reagents and the number of reactions that must take place. Just to exemplify the potential rewards of synthesizing new and effective antimicrobials, in 1995, the worldwide sales of erythromycin and its derivatives exceeded 3.5 billion dollars. This portion will examine the modifications of structure in the DEBS PKS to create new products in regards to erythromycin derivatives as well as completely new polyketides generated by various means of engineering the modular complex. There are five general methods in which DEBS is regularly modified: • Deletion or inactivation of active sites and modules • Substitution or addition of active sites and modules • Precursor-directed biosynthesis • KR replacement for altered stereospecificity • Tailoring enzyme modifications Deletion or inactivation of active sites and modules The first reported instance of genetic engineering of DEBS came in 1991 from the Katz group who deleted the activity of the KR in module 5 of DEBS which produced a 5-keto macrolide instead of the usual 5-hydroxy macrolide. Since then, deletion or inactivation (often via introduction of point mutations) of many active sites to skip reduction and/or dehydration reactions have been created. Such modifications target the various KR, DH, ER active sites seen on different modules in DEBS. In fact, whole modules can be deleted in order to reduce the chain-length of the polyketides and alter the cycle of reduction/dehydration normally seen. The activities of the two modules is identical, and the same erythromycin precursor (6-deoxyerythronolide B) was produced by the chimeric PKS; however, this shows the possibility of creating PKS with modules from two or even several different PKS in order to produce a multitude of new products. There is one problem with connecting heterologous modules though; there is recent evidence that the amino acid sequence between the ACP domain and the subsequent KS domain of downstream modules plays an important role in the transfer of the growing polyketide from one module to another. Ketoreductase replacement to alter stereospecificity In modular PKS, KR active sites catalyze stereospecific reduction of polyketides. Inversion of an alcohol stereocenter to the opposite stereoisomer is possible via replacement of a wild-type KR with a KR of the opposite specificity. Thus far, few attempts have been made to modify tailoring pathways, however, the enzymes which participate in such pathways are currently being characterized and are of great interest. Studies are facilitated by their respective genes being located adjacent to the PKS genes, and many are therefore readily identifiable. There is no doubt that in the future, alteration of tailoring enzymes could produce many new and effective antimicrobials. ==Structural studies==

As of late 2007, 8 structures have been solved for this class of enzymes, with PDB accession codes , , , , , , , and . Other names of this enzyme class is malonyl-CoA:propanoyl-CoA malonyltransferase (cyclizing). Other names in common use include erythronolide condensing enzyme, and malonyl-CoA:propionyl-CoA malonyltransferase (cyclizing). == References ==