Mutant Malonyl‐CoA Synthetases with Altered Specificity for Polyketide Synthase Extender Unit Generation

Koryakina, Irina; Williams, Gavin J.

doi:10.1002/cbic.201100383

Cited by 39 publications

(54 citation statements)

References 25 publications

(29 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This knowledge allowed in vitro precursor-directed biosynthesis of a fully extended and cyclized C2-ethyl narbonolide analog. Recently, engineered malonyl-CoA synthetases with expanded substrate specificity were created for the chemo-enzymatic synthesis of a broad panel of natural and non-natural extender units, ultimately leading to the discovery of promiscuity in a unique trans -acyltransferase [30•,31]. In addition, the terminal module and thioesterase domain of the 6-deoxyerythronolide B synthase (DEBS) was revealed to be remarkably tolerant to a range of extender units [32].…”

Section: Extender Unit Selection and Chain Elongationmentioning

confidence: 99%

Engineering polyketide synthases and nonribosomal peptide synthetases

Williams

2013

Current Opinion in Structural Biology

View full text Add to dashboard Cite

Naturally occurring polyketides and non-ribosomal peptides with broad and potent biological activities continue to inspire the discovery of new and improved analogs. The biosynthetic apparatus responsible for the construction of these natural products has been the target of intensive protein engineering efforts. Traditionally, engineering has focused on substituting individual enzymatic domains or entire modules with those of different building block specificity, or by deleting various enzymatic functions, in an attempt to generate analogs. This review highlights strategies based on site-directed mutagenesis of substrate binding pockets, semi-rational mutagenesis, and whole-gene random mutagenesis to engineer the substrate specificity, activity, and protein interactions of polyketide and non-ribosomal peptide biosynthetic machinery.

show abstract

Section: Extender Unit Selection and Chain Elongationmentioning

confidence: 99%

Engineering polyketide synthases and nonribosomal peptide synthetases

Williams

2013

Current Opinion in Structural Biology

View full text Add to dashboard Cite

show abstract

“…Each Ery6 AT variant (Figure 2C) was incubated with the diketide-SNAC thioester 3 and a 1:3 mixture of methylmalonyl-CoA ( 1 ) and propargylmalonyl-CoA ( 2 ) prepared via engineered MatB mutants 20, 29, 30 (see Supplemental Information for details). These conditions mimic desired in vivo feeding experiments whereby the non-natural extender unit is usually provided in excess over the natural substrate.…”

Section: Resultsmentioning

confidence: 99%

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering

et al. 2016

Self Cite

View full text Add to dashboard Cite

Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate towards a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.

show abstract

“…For the common compounds malonyl-CoA and methylmalonyl-CoA (28) the recently described methylmalonyl-CoA ligase MatB from Rhizobium trifolii is a reliable direct enzymatic synthesis route from free (methyl-)malonic acid (Route 6, [7,16]). Through active site mutagenesis, a more promiscuous MatB variant was created recently.…”

Section: α-Carboxylated Malonyl-coa Derivativesmentioning

confidence: 99%

“…Through active site mutagenesis, a more promiscuous MatB variant was created recently. This variant was used to generate other α-carboxylated (R)-malonyl-CoA derivatives in situ for in vitro polyketide synthesis assays [12,16]. Note however, that MatB synthesis is limited by commercial availablility of free malonic acid starting precursors.…”

Section: α-Carboxylated Malonyl-coa Derivativesmentioning

confidence: 99%

A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters

et al. 2016

View full text Add to dashboard Cite

Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short-to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.

show abstract

Mutant Malonyl‐CoA Synthetases with Altered Specificity for Polyketide Synthase Extender Unit Generation

Cited by 39 publications

References 25 publications

Engineering polyketide synthases and nonribosomal peptide synthetases

Engineering polyketide synthases and nonribosomal peptide synthetases

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering

A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters

Contact Info

Product

Resources

About