Molecular basis of Celmer's rules: role of the ketosynthase domain in epimerisation and demonstration that ketoreductase domains can have altered product specificity with unnatural substrates.

BACKGROUND: Polyketides are structurally diverse natural products with a range of medically useful activities. Non-aromatic bacterial polyketides are synthesised on modular polyketide synthase multienzymes (PKSs) in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function. RESULTS: We show that the ketoreductase (KR) domains of modules 5 and 6 of the erythromycin PKS, housed in the multienzyme subunit DEBS3, exert an unexpectedly low level of stereochemical control in reducing the keto group of a synthetic analogue of the diketide intermediate. This led us to construct a hybrid triketide synthase based on DEBS3 with ketosynthase domain ketosynthase (KS)5 replaced by the loading module and KS1. The construct in vivo produced two major triketide stereoisomers, one expected and one surprising. The latter was of opposite configuration at three out of the four chiral centres: the branching alkyl centre was that produced by KS1 and, surprisingly, both hydroxyl centres produced by the reduction steps carried out by KR5 and KR6 respectively. CONCLUSIONS: These results demonstrate that the epimerising activity associated with module 1 of the erythromycin PKS can be conferred on module 5 merely by transfer of the KS1 domain. Moreover, the normally precise stereochemical control observed in modular PKSs is lost when KR5 and KR6 are challenged by an unfamiliar substrate, which is much smaller than their natural substrates. This observation demonstrates that the stereochemistry of ketoreduction is not necessarily invariant for a given KR domain and underlines the need for mechanistic understanding in designing genetically engineered PKSs to produce novel products.

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase.

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.

Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension.

BACKGROUND: Polyketides are a large and structurally diverse group of natural products that include antibiotics, antifungal agents and immunosuppressant compounds. Polyketides are biosynthesised in filamentous bacteria on modular polyketide synthases (PKSs) in which each cycle of chain extension requires a different 'module' of enzymatic activities. The recently proposed dimeric model for modular PKSs predicts that even a single-module PKS should be catalytically active in the absence of other PKS components. Researchers are also interested in manipulating the stereochemical outcome of polyketide chain extension using genetic engineering of domains within each module. RESULTS: We have constructed a minimal modular PKS from the erythromycin-producing PKS (DEBS) of Saccharopolyspora erythraea. The diketide synthase (DKS1-2) consists of a single chimaeric extension module, derived from the DEBS module 1 ketoacyl-ACP synthase (KS), sandwiched between a loading module and a chain-terminating thioesterase. When DKS1-2 was expressed in S. erythraea, the strain preferentially6 accumulated the diketide (2R, 3S)-2-methyl-3-hydroxy pentanoic acid. CONCLUSIONS: These results demonstrate that, as predicted, even a single-module PKS is catalytically active in the absence of other DEBS proteins. In its normal context, the ketosynthase domain KS1 is thought to generate a (2S)-2methyl-3-hydroxy intermediate by epimerising the initial product of carbon-carbon chain formation, the (2R)-2-methyl-3-ketoester. The observed formation of the alternative (2R)-methyl-3-hydroxy product catalysed by DKS1-2 provides strong support for this proposal, and indicates how targeted alteration of stereospecificity can be achieved on a modular PKS.

FTIR study of the thermal denaturation of horseradish and cytochrome c peroxidases in D2O.

Fourier transform infrared (FTIR) spectroscopy was employed to examine the thermal denaturation of the Fe(III), Fe(II), and Fe(II)-CO forms of cytochrome c peroxidase and horseradish peroxidase in phosphate buffer at pD 7.0. The amide I' regions of the deconvolved spectra are consistent with predominantly alpha-helical secondary structure around room temperature, but the alpha-helical absorption of the two peroxidases differs significantly; bands assigned to alpha-helical components occur at 1659 and 1649 cm-1 in horseradish peroxidase and at 1652 and 1637 cm-1 in cytochrome c peroxidase. The thermal denaturation mechanisms of the peroxidases also vary. All three forms of cytochrome c peroxidase retain their secondary structure up to 50 degrees C, when bands characteristic of aggregation (1616 and 1684 cm-1) appear in the amide I' region, and above 55 degrees C rapid loss of secondary structure is accompanied by enhanced aggregation. In horseradish peroxidase, on the other hand, the Fe(III) and Fe(II) states exhibit dissimilar denaturation mechanisms. Slow, gradual alteration of secondary structure is observed for Fe(III) horseradish peroxidase on heating, and polypeptide unfolding appears to be complete around 90 degrees C, without aggregation. In Fe(II) and Fe(II)-CO horseradish peroxidase, aggregation bands appear at approximately 55 degrees C, signaling the onset of denaturation. Frequency shifts in the v(CO) bands above room temperature reveal the conformational changes in the heme cavity precede global conformational changes in cytochrome c peroxidase but not in horseradish peroxidase. The reduction in amide II intensities, due to peptide H-D exchange on heating the peroxidases in D2O, indicates the formation above room temperature of partially unfolded states with increased solvent accessibility but intact secondary structures.