Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases.

Autor:	Klaus M; From the Departments of Chemistry and Chemical Engineering and Stanford ChEM-H, Stanford University, Stanford, California 94305 and., Ostrowski MP; From the Departments of Chemistry and Chemical Engineering and Stanford ChEM-H, Stanford University, Stanford, California 94305 and., Austerjost J; From the Departments of Chemistry and Chemical Engineering and Stanford ChEM-H, Stanford University, Stanford, California 94305 and., Robbins T; From the Departments of Chemistry and Chemical Engineering and Stanford ChEM-H, Stanford University, Stanford, California 94305 and., Lowry B; From the Departments of Chemistry and Chemical Engineering and Stanford ChEM-H, Stanford University, Stanford, California 94305 and., Cane DE; the Department of Chemistry, Brown University, Providence, Rhode Island 02192-9108., Khosla C; From the Departments of Chemistry and Chemical Engineering and Stanford ChEM-H, Stanford University, Stanford, California 94305 and khosla@stanford.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2016 Jul 29; Vol. 291 (31), pp. 16404-15. Date of Electronic Publication: 2016 May 31.
DOI:	10.1074/jbc.M116.730531
Abstrakt:	The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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