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

• Published in: Biochimica et biophysica acta Vol. 1860; no. 3; pp. 486 - 497
• Main Authors: Argyropoulos, Panos, Bergeret, Fabien, Pardin, Christophe, Reimer, Janice M., Pinto, Atahualpa, Boddy, Christopher N., Schmeing, T. Martin
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2016
• Subjects: biosynthesis; biphenyl; Catalytic Domain; erythromycin; Erythromycin - analogs & derivatives; Erythromycin - biosynthesis; High-resolution structure; hydrolysis; Inhibition; Non-hydrolyzable acyl-enzyme intermediates; Polyketide synthase; polyketide synthases; Protein Structure, Tertiary; Substrate Specificity; therapeutics; Thioesterase; Thiolester Hydrolases - chemistry; transesterification; Polyketide synthase; High-resolution structure; Thioesterase; Inhibition; Non-hydrolyzable acyl-enzyme intermediates
• DOI: 10.1016/j.bbagen.2015.11.007
• ISSN: 0304-4165; 0006-3002

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. [Display omitted] •1.7Å resolution structure of a new construct of the erythromycin thioesterase•Diphenyl phosphonate inhibitors of the erythromycin thioesterases were synthesized and assayed.•2.1Å resolution structure of allylphosphonate adduct of the erythromycin thioesterase•Slow maturation of initial phosphonate-enzyme adduct limits the use of diphenyl phosphonate esters.