Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase
| Autor: | Iain R. Challis, Lindsey Low, Peter F. Leadlay, Rolf Müller, Barrie Wilkinson, Steven G. Kendrew, Jose Garcia-Bernardo, Hans-Jürgen Hecht, Hrvoje Petković, Axel Sandmann, Barbara Silakowski, Enej Kuščer, Nicola Beeston |
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| Rok vydání: | 2008 |
| Předmět: |
Stereochemistry
Amino Acid Motifs Molecular Sequence Data Heterologous Epothilone Biochemistry Substrate Specificity Lactones Protein structure Transferases polycyclic compounds medicine Humans Amino Acid Sequence Physical and Theoretical Chemistry Epothilone Polyketide Synthase Binding site Peptide sequence Binding Sites biology Chemistry Organic Chemistry Active site Protein Structure Tertiary Acyltransferase Mutagenesis Site-Directed biology.protein lipids (amino acids peptides and proteins) Polyketide Synthases Saccharopolyspora medicine.drug |
| Zdroj: | Org. Biomol. Chem.. 6:500-506 |
| ISSN: | 1477-0539 1477-0520 |
| DOI: | 10.1039/b714804f |
| Popis: | The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity. |
| Databáze: | OpenAIRE |
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