The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase

Abstract

The x-ray crystal structure of a 194-kDa fragment from module 5 of the 6-deoxyerythronolide B synthase has been solved at 2.7 Å resolution. Each subunit of the homodimeric protein contains a full-length ketosynthase (KS) and acyl transferase (AT) domain as well as three flanking ''linkers.'' The linkers are structurally well defined and contribute extensively to intersubunit or interdomain interactions, frequently by means of multiple highly conserved residues. The crystal structure also reveals that the active site residue Cys-199 of the KS domain is separated from the active site residue Ser-642 of the AT domain by Ϸ80 Å. This distance is too large to be covered simply by alternative positioning of a statically anchored, fully extended phosphopantetheine arm of the acyl carrier protein domain from module 5. Thus, substantial domain reorganization appears necessary for the acyl carrier protein to interact successively with both the AT and the KS domains of this prototypical polyketide synthase module. The 2.7-Å KS-AT structure is fully consistent with a recently reported lower resolution, 4.5-Å model of fatty acid synthase stucture, and emphasizes the close biochemical and structural similarity between polyketide synthase and fatty acid synthase enzymology. modular megasynthase ͉ multienzyme assembly ͉ polyketide synthase

Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli

“…2A). In the first approach, the PKS4 KSMAT didomain and the ACP domain were extracted as stand-alone proteins based on conserved domain boundaries (22). This was designed to mimic the dissociated components of bacterial type II minimal PKS.…”

Probing the interactions of an acyl carrier protein domain from the 6‐deoxyerythronolide B synthase

“…The subset of these residues with surface-exposed side chains was defined as binding sites for the purpose of developing a model for KS-ACP recognition. Using the crystal structure of [KS3][AT3] 12 and NMR structure of the ACP2 domain, 11 computational docking simulations guided by the experimental constraints were performed (see Experimental Procedures for details). 14,15 The resulting model (Fig.…”

“…For example, the mixture of [KS3][AT3] and malonyl-ACP2 (50 lM) was incubated at room temperature for up to 3 h and analyzed by radio-SDS-PAGE. 9,12 After purification by HiTrap-Q anion exchange, these proteins were subjected to several rounds of buffer exchange in NMR buffer (30 mM phosphate buffer, pH 7.0, 0.05% NaN 3 , and 10% D 2 O) at 4 C. Protein concentration was performed with a 30-kDa cutoff Centricon (Millipore). The purified proteins (final concentration 85 lM) were added to apo-or holo-ACP2 (final concentration 112 lM, also in 30 mM phosphate buffer, pH 7.0, 0.05% NaN 3 , and 10% D 2 O) on ice.…”

“…2) 11 and two structurally characterized [KS][AT] didomains from modules 3 and 5 of DEBS. 12,13 The latter proteins were chosen because earlier studies had revealed that ACP2 has a marked preference for module 3 over module 5 during intermodular chain translocation. 8 During the biosynthesis of erythromycin, the ACP2 domain collaborates with the enzymatic domains of module 2 to synthesize a highly labile triketide intermediate, which is then translocated onto KS3.…”

A phosphopantetheinylating polyketide synthase producing a linear polyene to initiate enediyne antitumor antibiotic biosynthesis

“…8). We prepared constructs expressing the individual ACP domain of SgcE, a general approach that has been successfully used to elucidate domain function for large multidomain PKS and FAS (39)(40)(41). Because nothing was known about the structural elements necessary for potential PPTase recognition of this type of ACP, five versions were prepared, ranging from a standard ACP size of 81 amino acids (ACP 81 ) to a long version (ACP 212 ) encompassing the majority of the region residing between the AT and KR.…”