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Catalytic modules of assembly-line polyketide synthases (PKSs) have previously been observed in two very different conformations-an "extended" architecture and an "arch-shaped" architecture-although the catalytic relevance of neither has been directly established. By the use of a fully human naïve antigen-binding fragment (Fab) library, a high-affinity antibody was identified that bound to the extended conformation of a PKS module, as verified by X-ray crystallography and tandem size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS). Kinetic analysis proved that this antibody-stabilized module conformation was fully competent for catalysis of intermodular polyketide chain translocation as well as intramodular polyketide chain elongation and functional group modification of a growing polyketide chain. Thus, the extended conformation of a PKS module is fully competent for all of its essential catalytic functions.

Various researches are conducted to study the X-ray crystal structure of aristolochene synthase from Aspergillus terreus, which helps in the catalysis of the cyclization of the universal sesquiterprene precursor, farnesyl diphosphate to give cyclic compounds. The study reveals that the trerpenoid cyclase active site plays a very important role in binding of the substrate, as it is highly evolvable in an [alpha]-helical fold.

The molecular genetics and biological experiments that provide evidence for the identity of the ptl gene cluster are illustrated. The second glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isozyme (gap2, SAV6296) is expressed in Escherichia coli and is inactivated by pentalenolactone.

The synthesis of 6-deoxyerythronolide B, the macrocyclic aglycone of the antibiotic erythromycin by a polyketide synthase (PKS) that has emerged as the prototypical modular megasynthase is described.

Investigation of the recombinant picromycin/methymycin synthase (PICS) module, 2+TE and its mutants establishes the structure and stereochemistry of the intermediates of the polyketide chain elongation cycle, catalyzed by a DH-containing module, that adds an equivalent of acetate and generates a new double bond. This also confirms the importance of key active site residues in both KR and DH domains.

The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP(sub i)) and aza analogues of the bisabolyl carbocation intermediate are reported. Structural analysis of the R340K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP(sub i) leaving group.

The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The rate constants for the individual biochemical steps are determined by a combination of protein acylation and trasthioesterification experiments.

The results of systematic steady-state kinetic analysis of a series of active site mutants of YgbP that elucidate the role key active residues and also describe pulse-chase experiments that establish the order of binding of the substrates of the enzyme catalyzed reaction are reported. In addition, Thr140, Arg109, Asp106, and Thr165 are shown to play critical roles in binding and proper orientation of the 2-C-methyl-D-erythritol 4-phosophate (MEP) substrate.

The transacylation of malonyl groups onto an acyl transferase (AT)-null form of a mutant modular polyketide synthase by malonyl-CoA:ACP transacylase is an effective strategy for the engineered biosynthesis of site specifically modified polyketides. The productivity of the modified system is comparable to that of the wild-type synthase in vitro and in vivo.

Picromycin synthase (PICS), a multifunctional, modular polyketide synthase (PKS) catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide. PICS modules were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products.

Antiparallel helical interactions between intermodular linkers play an important role in chain transfer of a growing polyketide chain. The linker regions are subjected to structural and mutational analysis in order to understand the mechanism of protein-protein recognition.

Research has been conducted on modular polyketide synthases' acyltransferase domains. The properties of some of these domains have been investigated, and the use of the expression system and assay formats in investigating the substrate specificity of the loading acyltransferase domain of 6-deoxy-erythronolide B synthase, methyl-malonyl-specific extender acyltransferase domain from 6-deoxy-erythronolide B synthase and a malonyl-specific extender acyltransferase domain from rapamycin synthase are described.

Research has been conducted on the modular polyketide synthases. The crystal structure of the synthases' forms and the effect of pH on the active site channel structure have been investigated and are discussed.

Research has been conducted on the methymycin/picromycin synthase' thioesterase domain. The heterologous expression of this domain has been carried out and the role of the active site residues has been investigated via the use of site-directed mutagenesis.

Research proposes a mechanism for the active site peptide loop conformational change, in pyridoxine 5(sup)' -phosphate synthase, by the substrates so as to provide access to the active site, whose residues in turn rearrange for catalysis and pyridoxine 5'-phosphate formation.

Research illustrates the active site residue modification by site-directed mutagenesis to elucidate the mechanism of action of pentalenene synthase. Active site base histidine, aspartates in the aspartate-rich domain, and aspargine-219 mutagenesis is described.

Results reveal that linker interactions and acyl carrier protein-ketosynthase interactions equally contribute to the module 2-module 3 and the module-4-module 5 interfaces in 6-deoxyerythronolide B synthase. However, nonnatural acyl carrier protein domains presented substrates are accepted by module 2 and 6, which is exploited in the engineering of hybrid polyketide synthases.

Research has been conducted on D100E trichodiene synthase. The crystal structure of this synthase have been examined both in the unliganded state and in complex with the reaction product pyrophosphate and the details are reported.

The precursor-directed biosynthesis of 16-membered macrolides by erythromycin polyketide synthases is described.

The C- and N-terminal ends of polypeptides in polyketide synthases contain short peptides that facilitate the transfer of intermediates between nonconsecutive modules. These 20- to 40-residue peptides must be considered when creating synthetic versions of the enzyme.

A study that is an attractive prototype for the expression, analysis and engineering of acyltransferase domains in modular polyketide synthases has been carried out to look at substrate specificity of the loading didomain of the erythromycin polyketide synthase. The loading didomain of 6-deoxyerythronolide B synthase (DEBS) can accept a broad range of substrates. Sfp was found to efficiently modify the acyl carrier protein (ACP(sub.L)) domain of the acyltransferase (AT(sub.L))-ACP(sub.L) protein with phosphopantetheine in vivo. The holo-enzyme that resulted was tested against various substrates to study its ability to discriminate between structurally different primer units.

The substrate specificity of modules derived from 6-Deoxyertythronolide B synthase (DEBS) are analyzed. The observed high degree of similarity in the specificity of the modules was tested. Also observed was a distinct preference by the modules for syn diketides over anti diketides. The results are analyzed from the perspective of an evolutionary and protein engineering

The role of the acyltransferase (AT) domains of modular polyketide synthases in the choice and stereochemical fate of extender units was examined by using domain mutagenesis. The results showed the interchangeability of the modular polyketide synthase 6-deoxyerythronolide B synthase (DEBS) AT4 with either the AT2 or AT1 domain in formation of AT4. This finding suggests that no intrinsic spimerase activity is associated with any of the three AT domains. It is also suggested that the epimerization of the (2S)-methylmalonyl-CoA extender units is not affected by the domains of DEBS.

This article presents a summary of reviews of polyketide synthesases along with implications for biosynthetic engineering. Parallels to fatty acid biosynthesis, the partial processing of intermediates, and the possibility of reprogramming biosynthetic pathways are discussed.

6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase that catalizes the stepwise repetitive condensation of complex products from simple carboxylic acid building blocks. The terminal module of DEBS, DEBS3, is competent in the biosynthesis of polyketide even in the absence of the other modules. A tricarboxylic acid can be used to prime this module and it undertakes two condensation cycles that ultimately produce 6-deoxyerythronolide.

The functional orientation of the acyltransferase (AT) domain was examined by inactivating the AT domain of module2 through site-directed mutagenesis utilizing a bimodular derivative of the 6-deoxyerythronolide B synthase. Both the inactivated AT domain of module 2 polypeptide and the inactive ketosynthase protein were found to promote polyketide synthesis, indicating that AT2 may function from either subunit. The results further indicated that the two clusters of active sites may simultaneously use the AT domain within the same dimeric module.