Solution Structure of an Acyl Carrier Protein Domain from a Fungal Type I Polyketide Synthase,

Abstract

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.

The structural organization of substrate loading in iterative polyketide synthases

“…6b) and shares a higher sequence identity (a CP2: 40% vs . ACP1: 30%) with the previously characterized PksA ACP 20 , 29 . For crosslinking, the loaded ACP2 was added to a variant of CTB1 SAT-KS-MAT in which the SAT and MAT active sites were mutated to strictly direct crosslinking to the KS (denoted as SAT°-KS-MAT°).…”

Substrate Placement Influences Reactivity in Non-heme Fe(II) Halogenases and Hydroxylases

“…PPant structures selected from a NMR ensemble of an acyl carrier protein (Protein Data Bank code 2KR5) (7) were docked inside a model of SyrB2 (Protein Data Bank code 2FCT) (11) using the Glide software package, and the complex was solvated with water (34). The pK a values of ionizable side chains for the protein were calculated in the environment of each surrounding residue and assigned for a pH of 7.4 (35-37).…”

“…Nonribosomal peptide and polyketide synthases (3,4), which synthesize clinically important natural products (5,6), can associate with tailoring enzymes, which act on small molecule substrates that are delivered by a phosphopantetheine (PPant) 3 tether bound to a carrier protein ( Fig. 1) (4,(7)(8)(9). The PPant-dependent halogenase, SyrB2 (10,11), chlorinates the methyl group of tethered L-Thr as part of the biosynthetic pathway for syringomycin E, an antifungal antibiotic that acts by forming channels in lipid bilayers (10,12).…”

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

“…Polyketides produced from NRPKSs are aromatic compounds with sizes ranging from monocyclic aromatics to those containing multiple fused rings. Starting from the N terminus of the megasynthase, the following domains are typically found in an NRPKS: a starter unit:ACP transacylase (SAT) that selects the starter unit (13); a ketosynthase (KS) that catalyzes repeated decarboxylative condensation to elongate the polyketide backbone; a malonyl-CoA:ACP transacylase (MAT) that selects and transfers the extender unit malonyl-CoA; a product template (PT) domain that controls the immediate cyclization regioselectivity of the reactive polyketide backbone (14); an acyl-carrier protein (ACP) that serves as the tether of the growing and completed polyketide via its phosphopantetheinyl arm (15); and a thioesterase/Claisen-like cyclase (TE/CLC) domain that cyclizes and releases the product (16). Other domains such as methyltransferase (MT) and reductase (R) can be found in subclades of the NRPKSs (6).…”