Insights into modular polyketide synthase loops aided by repetitive sequences.

The loops of modular polyketide synthases (PKSs) serve diverse functions but are largely uncharacterized. They frequently contain amino acid repeats resulting from genetic events such as slipped-strand mispairing. Determining the tolerance of loops to amino acid changes would aid in understanding and engineering these multidomain molecule factories. Here, tandem repeats in the DNA encoding 949 modules within 129 cis-acyltransferase PKSs were cataloged, and the locations of the corresponding amino acids within the module were identified. The most frequently inserted interdomain loop corresponds with the updated module boundary immediately downstream of the ketosynthase (KS), while the loops bordering the dehydratase are nearly intolerant to such insertions. From the 949 modules, no repetitive sequence loop insertions are located within ACP, and only 2 reside within KS, indicating the sensitivity of these domains to alteration.

A soybean acyl carrier protein, GmACP, is important for root nodule symbiosis.

Legumes (members of family Fabaceae) establish a symbiotic relationship with nitrogen-fixing soil bacteria (rhizobia) to overcome nitrogen source limitation. Single root hair epidermal cells serve as the entry point for bacteria to infect the host root, leading to development of a new organ, the nodule, which the bacteria colonize. In the present study, the putative role of a soybean acyl carrier protein (ACP), GmACP (Glyma18g47950), was examined in nodulation. ACP represent an essential cofactor protein in fatty acid biosynthesis. Phylogenetic analysis of plant ACP protein sequences showed that GmACP was classified in a legume-specific clade. Quantitative reverse-transcription polymerase chain reaction analysis demonstrated that GmACP was expressed in all soybean tissues but showed higher transcript accumulation in nodule tissue. RNA interference-mediated gene silencing of GmACP resulted in a significant reduction in nodule numbers on soybean transgenic roots. Fluorescent protein-labeled GmACP was localized to plastids in planta, the site of de novo fatty acid biosynthesis in plants. Analysis of the fatty acid content of root tissue silenced for GmACP expression, as determined by gas chromatography-mass spectrometry, showed an approximately 22% reduction, specifically in palmitic and stearic acid. Taken together, our data provide evidence that GmACP plays an important role in nodulation.

Acyl carrier protein structural classification and normal mode analysis.

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well.

Type I polyketide synthases that require discrete acyltransferases.

The diverse structures of polyketide natural products are reflected by the equally diverse polyketide biosynthetic enzymes, namely polyketide synthases (PKSs). Three major classes of PKSs are known-noniterative type I PKSs, iterative type II PKSs and acyl carrier protein-independent type III PKSs, each of which consists of additional variants. One such variant is the noniterative type I PKS in which each PKS module lacks the cognate acyltransferase (AT) domain. The essential AT activity is instead provided by a discrete AT in trans. Termed "AT-less" type I PKSs, the loading of the malonate extender units by the discrete AT enzyme LnmG to each of the AT-less PKS modules of LnmI and LnmJ was confirmed experimentally for biosynthesis of the anticancer antibiotic leinamycin (LNM). The LNM PKS has since served as a model for the continuous discovery of numerous additional AT-less type I PKSs incorporating variable extender units. However, biochemical characterization of AT-less type I PKSs remains very limited, and the mechanism by which AT-less type I PKSs accommodate multiple extender units is unknown. This chapter provides the protocols used to establish and characterize the LNM PKS. Application of these methods to other AT-less type I PKSs should aid the biochemical characterization and hence possible exploitation of these unique PKSs for polyketide natural product structural diversity by combinatorial biosynthetic methods.

Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family.

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel alpha helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its "recognition" helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein.

We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.