Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units Michelle L Heathcote,

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Presentation transcript:

Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units Michelle L Heathcote, James Staunton, Peter F Leadlay Chemistry & Biology Volume 8, Issue 2, Pages 207-220 (February 2001) DOI: 10.1016/S1074-5521(01)00002-3

Fig. 1 Chain release catalysed by TE domains in (a) FAS and (b) PKS systems. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 2 Mechanism of action of TEs to form an acyl–enzyme intermediate. The resultant tetrahedral intermediate is released by nucleophilic attack of an external nucleophile or an internal nucleophile to form a lactone. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 3 (a) A typical decarboxylative chain elongation step in FASs and PKSs. (b) Aberrant decarboxylation by the KS domain to give an acetate, propionate or butyrate residue attached to the ACP. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 4 Purification of the heterologously expressed tylosin TE II domain. Samples of the recombinant protein were analysed by SDS–PAGE at different stages of a typical purification procedure. Gels were visualised by staining with Coomassie brilliant blue. Lane 1, molecular weight markers; lane 2, soluble fraction from induced cells carrying pMLH27; lane 3, Ni-NTA column flow-through; lane 4, purified tylosin TE II after affinity chromatography; lane 5, molecular weight markers; lane 6, purified tylosin TE II after anion exchange; lane 7, contaminant proteins eluted after anion exchange. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 5 ESI-MS spectrum of the recombinant tylosin TE II protein. The expected molecular mass of the enzyme from which the N-terminal methionine has been removed is [M+H]+, 29 679.4 Da. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 6 Substrates chosen for investigation of the specificity of tylosin TE II in vitro. (a) Substrates 1–8 were designed to test the effect of chain length on TE II specificity. (b) The ketide analogues 9 and 10 were modelled on the diketide 11 and triketide 12 biosynthetic intermediates of the tylosin PKS. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 7 Rate versus concentration plots for the hydrolysis of acyl NAC thioester substrates 1–4 and 9 by recombinant tylosin TE II. The kcat/KM values for substrates 1, 4 and 9 were calculated from the linear slope of each plot at low substrate concentration, whereas the kcat/KM values for substrates for substrates 2 and 3 were estimated using a Michaelis–Menten curve fit (Kaleidagraph software) or using an Eadie–Hofstee plot. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 8 Rate versus concentration plots for the hydrolysis of acyl p-nitrophenyl substrates 5–8 and 10 by recombinant tylosin TE II. The kcat/KM for each substrate was calculated from the linear slope of each plot at low substrate concentration. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 9 Analysis of acylation experiments with tylosin TE II and p-nitrophenyl propionate 6 by ESI-MS. (a) Acylated tylosin TE II (with His6 tag). (b) Acylated tylosin TE II (no His6 tag). (c) Tylosin TE II (no His6 tag)+DTNB in the absence of p-nitrophenyl propionate 6. (d) Acylated tylosin TE II (no His6 tag)+DTNB. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)

Fig. 10 A three-dimensional representation of a typical type I modular PKS multienzyme, based on the helical model. The complementary polypeptide chains are coloured pink and blue, with the enzymatic domains shown as spheres. The arrows show the independent paths of the two growing polyketide chains. In this arrangement, the KS of one polypeptide chain can only interact with the ACP of the opposing chain, as shown. If blockage of one of the ACP domains occurs, the rate of passage of the polyketide chain through that module will be halved. Chemistry & Biology 2001 8, 207-220DOI: (10.1016/S1074-5521(01)00002-3)