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Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase.
Biochemistry. 2004 Nov 09; 43(44):13892-8.B

Abstract

The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.

Authors+Show Affiliations

Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15518537

Citation

Kim, Chu-Young, et al. "Reconstituting Modular Activity From Separated Domains of 6-deoxyerythronolide B Synthase." Biochemistry, vol. 43, no. 44, 2004, pp. 13892-8.
Kim CY, Alekseyev VY, Chen AY, et al. Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase. Biochemistry. 2004;43(44):13892-8.
Kim, C. Y., Alekseyev, V. Y., Chen, A. Y., Tang, Y., Cane, D. E., & Khosla, C. (2004). Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase. Biochemistry, 43(44), 13892-8.
Kim CY, et al. Reconstituting Modular Activity From Separated Domains of 6-deoxyerythronolide B Synthase. Biochemistry. 2004 Nov 9;43(44):13892-8. PubMed PMID: 15518537.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase. AU - Kim,Chu-Young, AU - Alekseyev,Viktor Y, AU - Chen,Alice Y, AU - Tang,Yinyan, AU - Cane,David E, AU - Khosla,Chaitan, PY - 2004/11/3/pubmed PY - 2005/1/12/medline PY - 2004/11/3/entrez SP - 13892 EP - 8 JF - Biochemistry JO - Biochemistry VL - 43 IS - 44 N2 - The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15518537/Reconstituting_modular_activity_from_separated_domains_of_6_deoxyerythronolide_B_synthase_ L2 - https://dx.doi.org/10.1021/bi048418n DB - PRIME DP - Unbound Medicine ER -