Crystal Structure of the Macrocycle-forming Thioesterase Domain of the Erythromycin Polyketide Synthase: Versatility from a Unique Substrate Channel

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2001 Dec 26

PMID 11752428

Citations 69

Authors

S C Tsai

L J Miercke

J Krucinski

R Gokhale

J C Chen

P G Foster

D E Cane

C Khosla

R M Stroud

Affiliations

Soon will be listed here.

Abstract

As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-A resolution. Its overall tertiary architecture belongs to the alpha/beta-hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dimer interface and a substrate channel that passes through the entire protein. The active site triad, comprised of Asp-169, His-259, and Ser-142, is located in the middle of the substrate channel, suggesting the passage of the substrate through the protein. Modeling indicates that the active site can accommodate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explain the observed substrate specificity of this TE and offer strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the 2-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangement of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular polyketide synthases have a similar fold, dimer 2-fold axis, and substrate channel geometry.

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References

Gokhale R, Hunziker D, Cane D, Khosla C . Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase. Chem Biol. 1999; 6(2):117-25. DOI: 10.1016/S1074-5521(99)80008-8. View

McDaniel R, Thamchaipenet A, Gustafsson C, Fu H, Betlach M, Ashley G . Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel "unnatural" natural products. Proc Natl Acad Sci U S A. 1999; 96(5):1846-51. PMC: 26699. DOI: 10.1073/pnas.96.5.1846. View

Nardini M, Dijkstra B . Alpha/beta hydrolase fold enzymes: the family keeps growing. Curr Opin Struct Biol. 1999; 9(6):732-7. DOI: 10.1016/s0959-440x(99)00037-8. View

Bellizzi 3rd J, Widom J, Kemp C, Lu J, Das A, Hofmann S . The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis. Proc Natl Acad Sci U S A. 2000; 97(9):4573-8. PMC: 18274. DOI: 10.1073/pnas.080508097. View

Li J, Derewenda U, Dauter Z, Smith S, Derewenda Z . Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme. Nat Struct Biol. 2000; 7(7):555-9. DOI: 10.1038/76776. View

Devedjiev Y, Dauter Z, Kuznetsov S, Jones T, Derewenda Z . Crystal structure of the human acyl protein thioesterase I from a single X-ray data set to 1.5 A. Structure. 2000; 8(11):1137-46. DOI: 10.1016/s0969-2126(00)00529-3. View

Winn M, Isupov M, Murshudov G . Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr D Biol Crystallogr. 2001; 57(Pt 1):122-33. DOI: 10.1107/s0907444900014736. View

Rowe C, Bohm I, Thomas I, Wilkinson B, Rudd B, Foster G . Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase. Chem Biol. 2001; 8(5):475-85. DOI: 10.1016/s1074-5521(01)00024-2. View

Wu N, Tsuji S, Cane D, Khosla C . Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. J Am Chem Soc. 2001; 123(27):6465-74. DOI: 10.1021/ja010219t. View

10.

Donadio S, Staver M, McAlpine J, Swanson S, Katz L . Modular organization of genes required for complex polyketide biosynthesis. Science. 1991; 252(5006):675-9. DOI: 10.1126/science.2024119. View

11.

Otwinowski Z, Minor W . Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997; 276:307-26. DOI: 10.1016/S0076-6879(97)76066-X. View

12.

Corpet F . Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988; 16(22):10881-90. PMC: 338945. DOI: 10.1093/nar/16.22.10881. View

13.

Holak T, Kearsley S, Kim Y, Prestegard J . Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations. Biochemistry. 1988; 27(16):6135-42. DOI: 10.1021/bi00416a046. View

14.

Matthews B . Determination of protein molecular weight, hydration, and packing from crystal density. Methods Enzymol. 1985; 114:176-87. DOI: 10.1016/0076-6879(85)14018-8. View

15.

Van Duyne G, Standaert R, Karplus P, Schreiber S, Clardy J . Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J Mol Biol. 1993; 229(1):105-24. DOI: 10.1006/jmbi.1993.1012. View

16.

Cortes J, Wiesmann K, Roberts G, Brown M, Staunton J, Leadlay P . Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage. Science. 1995; 268(5216):1487-9. DOI: 10.1126/science.7770773. View

17.

Lawson D, Derewenda U, Serre L, Ferri S, Szittner R, Wei Y . Structure of a myristoyl-ACP-specific thioesterase from Vibrio harveyi. Biochemistry. 1994; 33(32):9382-8. DOI: 10.1021/bi00198a003. View

18.

Crump M, Crosby J, Dempsey C, Parkinson J, Murray M, Hopwood D . Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2). Biochemistry. 1997; 36(20):6000-8. DOI: 10.1021/bi970006+. View

19.

Jacobsen J, Hutchinson C, Cane D, Khosla C . Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science. 1997; 277(5324):367-9. DOI: 10.1126/science.277.5324.367. View

20.

Schrag J, Cygler M . Lipases and alpha/beta hydrolase fold. Methods Enzymol. 1997; 284:85-107. DOI: 10.1016/s0076-6879(97)84006-2. View