No abstract

Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules.

Acyl (peptidyl) carrier protein (ACP or PCP) is a crucial component involved in the transfer of thiol ester-bound intermediates during the biosynthesis of primary and secondary metabolites such as fatty acids, polyketides, and nonribosomal peptides. Although many carrier protein three-dimensional structures have been determined, to date there is no model available for a fungal type I polyketide synthase ACP. Here we report the solution structure of the norsolorinic acid synthase (NSAS) holo ACP domain that has been excised from the full-length multifunctional enzyme. NSAS ACP shows similarities in three-dimensional structure with other type I and type II ACPs, consisting of a four-helix bundle with helices I, II, and IV arranged in parallel. The N-terminus of helix III, however, is unusually hydrophobic, and Phe1768 and Leu1770 pack well with the core of the protein. The result is that unlike other carrier proteins, helix III lies almost perpendicular to the three major helices. Helix III is well-defined by numerous NMR-derived distance restraints and may be less flexible than counterparts in type II FAS and PKS ACPs. When the holo ACP is derivatized with a hexanoyl group, only minor changes are observed between the HSQC spectra of the two ACP species and no NOEs are observed for this hydrophobic acyl group. Along with the mammalian type I FAS, this further strengthens the view that type I ACPs do not show any significant affinity for hydrophobic (nonpolar) chain assembly intermediates attached via the 4'-phosphopantetheine prosthetic group.

Placental development and imprinting co-evolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes, IGF2 and IGF2R, code for the growth promoter insulin-like growth factor 2 and its binding inhibitor, mannose 6-phosphate/IGF2 receptor, respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes that lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that evolution of this ESE led to the fortuitous acquisition of M6P/IGF2R IGF2 binding that drew IGF2R into parental conflict prior to imprinting, that may have accelerated subsequent affinity maturation. † The sequence of molecular evolutionary events that established placental viviparity, genomic imprinting and parental conflict in mammals remain poorly understood (1) . Genomic imprinting occurs when expression of one allele of a diploid gene is silenced depending on the parent-of-origin, e.g. either from the father or the mother. Parental conflict over the distribution of resources to offspring has been supported by the observation of reciprocal imprinting of genes coding for the growth promoter Insulin-like growth factor 2 (IGF2), and the cation-independent mannose 6-phosphate/ IGF2 receptor (M6P/IGF2R or IGF2R) (2) . IGF2 and IGF2R are two of the approximately 80 genes imprinted in mammals, and two of the five genes (with INS, MEST/PEG1 and PEG10) imprinted in marsupials. So far, no evidence supports the existence of imprinting in monotremes despite the presence of a chorio-vitelline placenta (3, 4). On the basis of functional data, IGF2R transports M6P modified acid hydrolases to the pre-lysosomes (5). Of the 15 extra-cellular domains of IGF2R, domain 11 binds IGF2 in therians, and internalizes the ligand for degradation, whereas M6P bind to domains 3, 5 and 9 (5). Igf2 rescues placental dependent embryonic lethality associated with laboratory created murine parthenogenesis, implicating IGF2 supply as a regulator of placental development (6). Disruption of the maternal Igf2r allele results in Igf2 dependent overgrowth and fatality, supporting that IGF2R antagonizes the function of IGF2 (7,8). The structure of the unbound human domain 11 shows that the IGF2 binding site composed of defined loops (AB, CD, FG and HI, Fig. 1A and Fig. S1) but how this domain 11 evolved to bind IGF2, and the relationship to imprinting co-evolution, remain unknown (9-12).We established a high resolution structure of the human IGF2R:IGF2 complex and then compared this to other phylogenetically informative vertebrates. We adopted an NMR approach as the side chain amino acid interactions across the binding interface were not resolved in our 4.1Å resolution co-crystal structures (9). Wild-type human domain 11 and IGF2 failed to form a stable association in initial NMR studies. However, we ...

It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis.