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.

Biosynthesis of the Myxobacterial Antibiotic Corallopyronin A

“…[33] Conserved regions include the active site motif GHSXG, [39] which in the case of CorA is GHSLG ( Figure 2). Approximately 100 residues downstream of the active site serine, the YASH [40,41] or HAFH [39] motifs are usually regarded as indicative of methyl malonate or malonate activation, respectively. In the case of CorA, an AAFH sequence can be found, including the most significant phenylalanine residue (Figure 2), thus suggesting that only malonyl-CoA is used as an extender unit.…”

Isolation and Biosynthetic Analysis of Haliamide, a New PKS-NRPS Hybrid Metabolite from the Marine Myxobacterium Haliangium ochraceum

“…Such inconsistencies are not common in the typical bacterial PKS biosynthesis but sometimes observed in myxobacteria. We noticed that the AT domain of module 4 is phylogenetically close to the AT domain of EpoC (Supplementary Materials, Figure S4), a promiscuous AT loading both malonate and methylmalonate in the epothilone biosynthetic pathway from terrestrial myxobacterium Sorangium cellulosum [22].…”

“…Such inconsistencies are not common in the typical bacterial PKS biosynthesis but sometimes observed in myxobacteria. We noticed that the AT domain of module 4 is phylogenetically close to the AT domain of EpoC (Supplementary Materials, Figure S4), a promiscuous AT loading both malonate and methylmalonate in the epothilone biosynthetic pathway from terrestrial myxobacterium Sorangium cellulosum [22]. After integrating the last biosynthetic block (acetate unit) by module 5, the biosynthesis undergoes a particular chain termination by decarboxylation, leading to the formation of a terminal olefin of the haliamide molecule, which may be mediated by the sulfotransferase (ST)-thioesterase (TE) domains encoded in the C-terminus of HlaB (Figure 4b).…”

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

“…These domain swaps led to the identification of a geldanamycin analogue with fourfold higher affinity towards Hsp90, a protein drug target [70]. Domain swaps have been successfully implemented with other PKSs as well [59,64,71]. Despite the apparent success of AT domain swaps in producing novel polyketide products, these successes often come at the cost of decreased product titres [72], and some AT domains appear to be especially difficult to replace [58].…”