Towards a characterization of the structural determinants of specificity in the macrocyclizing thioesterase for deoxyerythronolide B biosynthesis.

Type I polyketide synthases (PKSs) are giant multidomain proteins that synthesize many therapeutics and other natural products. The synthesis proceeds by a thiotemplate mechanism whereby intermediates are covalently attached to the PKS. The release of the final polyketide is catalyzed by the terminal thioesterase (TE) domain through hydrolysis, transesterification, or macrocyclization. The PKS 6-deoxyerythronolide B synthase (DEBS) produces the 14-membered macrolide core of the clinically important antibiotic erythromycin. The TE domain of DEBS (DEBS TE) has well-established, empirically-defined specificities for hydrolysis or macrocyclization of native and modified substrates. We present efforts towards understanding the structural basis for the specificity of the thioesterase reaction in DEBS TE using a set of novel diphenyl alkylphosphonates, which mimic substrates that are specifically cyclized or hydrolyzed by DEBS TE. We have determined structures of a new construct of DEBS TE alone at 1.7Å, and DEBS TE bound with a simple allylphosphonate at 2.1Å resolution. Other, more complex diphenyl alkylphosphonates inhibit DEBS TE, but we were unable to visualize these faithful cyclization analogs in complex with DEBS TE. This work represents a first step towards using DEBS TE complexed with sophisticated substrate analogs to decipher the specificity determinants in this important reaction.

Liquid Crystalline Esters of Dibenzophenazines.

A series of esters of 2,3,6,7-tetrakis(hexyloxy)dibenzo[a,c]phenazine-11-carboxylic acid was prepared in order to probe the effects of the ester groups on the liquid crystalline behavior. These compounds exhibit columnar hexagonal phases over broad temperature ranges. Variations in chain length, branching, terminal groups, and the presence of cyclic groups were found to modify transition temperatures without substantially destabilizing the mesophase range.

Biosynthesis of ebelactone A: isotopic tracer, advanced precursor and genetic studies reveal a thioesterase-independent cyclization to give a polyketide ß-lactone.

Macrocyclization of polyketides generates arrays of molecular architectures that are directly linked to biological activities. The four-membered ring in oxetanones (ß-lactones) is found in a variety of bioactive polyketides (for example, lipstatin, hymeglusin and ebelactone), yet details of its molecular assembly have not been extensively elucidated. Using ebelactone as a model system, and its producer Streptomyces aburaviensis ATCC 31860, labeling with sodium [1-(13)C,(18)O2]propionate afforded ebelactone A that contains (18)O at all oxygen sites. The pattern of (13)C-(18)O bond retention defines the steps for ebelactone biosynthesis, and demonstrates that ß-lactone ring formation occurs by attack of a ß-hydroxy group onto the carbonyl moiety of an acyclic precursor. Reaction of ebelactone A with N-acetylcysteamine (NAC) gives the ß-hydroxyacyl thioester, which cyclizes quantitatively to give ebelactone A in aqueous ethanol. The putative gene cluster encoding the polyketide synthase (PKS) for biosynthesis of 1 was also identified; notably the ebelactone PKS lacks a terminal thioesterase (TE) domain and no stand alone TE was found. Thus the formation of ebelactone is not TE dependent, supporting the hypothesis that cyclization occurs on the PKS surface in a process that is modeled by the chemical cyclization of the NAC thioester.