title: Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase.
abstract: 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.
authors.affiliation: Biotica, Chesterford Research Park, Little Chesterford, Saffron Walden, Essex CB10 1XL, UK.
authors.forename: Hrvoje
authors.initials: H
authors.lastname: Petković
authors.forename: Axel
authors.initials: A
authors.lastname: Sandmann
authors.forename: Iain R
authors.initials: IR
authors.lastname: Challis
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Hecht
authors.forename: Barbara
authors.initials: B
authors.lastname: Silakowski
authors.forename: Lindsey
authors.initials: L
authors.lastname: Low
authors.forename: Nicola
authors.initials: N
authors.lastname: Beeston
authors.forename: Enej
authors.initials: E
authors.lastname: Kuscer
authors.forename: Jose
authors.initials: J
authors.lastname: Garcia-Bernardo
authors.forename: Peter F
authors.initials: PF
authors.lastname: Leadlay
authors.forename: Steven G
authors.initials: SG
authors.lastname: Kendrew
authors.forename: Barrie
authors.initials: B
authors.lastname: Wilkinson
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D007783:Lactones / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016930:Saccharopolyspora / Q000201:enzymology; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D007783:Lactones; D048630:Polyketide Synthases; D014166:Transferases